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WO2004018700A2 - Procede pour moduler ou identifier les agents qui modulent le calcium intracellulaire - Google Patents

Procede pour moduler ou identifier les agents qui modulent le calcium intracellulaire Download PDF

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Publication number
WO2004018700A2
WO2004018700A2 PCT/US2003/026409 US0326409W WO2004018700A2 WO 2004018700 A2 WO2004018700 A2 WO 2004018700A2 US 0326409 W US0326409 W US 0326409W WO 2004018700 A2 WO2004018700 A2 WO 2004018700A2
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cell
protein
test
calcium
seq
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PCT/US2003/026409
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WO2004018700A3 (fr
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Jack Roos
Gonul Velicelebi
Kenneth Stauderman
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Neurogenetics, Inc.
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Priority to AU2003265607A priority Critical patent/AU2003265607A1/en
Publication of WO2004018700A2 publication Critical patent/WO2004018700A2/fr
Publication of WO2004018700A3 publication Critical patent/WO2004018700A3/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6872—Intracellular protein regulatory factors and their receptors, e.g. including ion channels
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/04—Screening involving studying the effect of compounds C directly on molecule A (e.g. C are potential ligands for a receptor A, or potential substrates for an enzyme A)

Definitions

  • Methods of identifying agents that modulate intracellular calcium are provided. These include methods of screening for candidate intracellular calcium- modulating proteins and nucleic acids encoding such proteins and methods of identifying intracellular calcium-modulating proteins and nucleic acids encoding such proteins. Also provided are methods of modulating calcium within cells and treating disease by modulating intracellular calcium. BACKGROUND OF THE INVENTION
  • calcium is a key element in the transduction of signals into and within cells.
  • Cellular responses to growth factors, neurotransmitters, hormones and a variety of other signal molecules are initiated through calcium-dependent processes.
  • Many proteins are activated by binding calcium and in turn affect other proteins in signal cascade mechanisms in cells.
  • the normal basal concentration of free calcium in the cytoplasm of cells is about 50-100 nM whereas the extracellular calcium concentration is typically about 2 mM. Therefore, intracellular calcium levels and fluctuations thereof are tightly regulated by cells.
  • Calcium regulation by cells is accomplished through a variety of mechanisms, some of which are associated with particular cell types.
  • excitable cells such as muscle and nerve cells in which calcium signals are essential to functions including contraction and transmission of nerve impulses, contain voltage-gated calcium channels spanning the cell membrane. These channels respond to depolarization of the potential difference across the membrane and can open to permit an influx of calcium from the extracellular medium and a rapid increase in intracellular calcium concentrations.
  • Nonexcitable cells e.g., blood cells, fibroblasts and epithelial cells, as well as many excitable cells, contain channels that span intracellular membranes and that can open to permit an influx of calcium into the cytoplasm from calcium-storing organelles, such as the endoplasmic reticulum.
  • One such intracellular ion channel is the inositol 1 ,4,5-triphosphate (IP 3 ) receptor located in the membrane of the endoplasmic reticulum.
  • IP 3 receptor functions as a ligand-gated ion channel that permits passage of calcium upon binding of IP 3 released through hydrolysis of membrane phospholipids by activated phospholipase C (PLC).
  • PLC can be activated through agonist binding to a surface membrane G protein-coupled receptor. Activation of the IP 3 receptor results in the release of calcium stored in the endoplasmic reticulum into the cytoplasm. Reduced endoplasmic reticulum calcium concentration resulting from release of calcium therefrom provides a signal for influx of calcium from the extracellular medium into the cell. It appears that this influx of calcium does not rely on voltage-gated plasma membrane channels and does not involve activation of calcium channels by calcium. This calcium influx mechanism has been referred to as capacitative calcium entry (CCE) or store-operated calcium entry. The actual factor that directly activates influx of calcium across the plasma membrane in CCE is unknown, as is the identity of the molecule or molecules that provide for mobilization of calcium across the plasma membrane and into the cell.
  • CCE capacitative calcium entry
  • identifying agents that modulate calcium levels are provided herein. Also provided herein are methods of identifying candidate agents for use in treating or preventing a disease or disorder and methods of treating a subject having a disease or disorder by administering to the subject an agent that modulates intracellular calcium. Also provided are methods of modulating lipid transport and/or storage in a cell by altering calcium levels in a cell.
  • agents that modulate intracellular calcium levels are identified.
  • Agents that modulate intracellular calcium levels may be identified by evaluating and comparing calcium levels in a cell having one or more proteins involved in calcium homeostasis in the presence and absence of a test agent.
  • calcium levels of the cell and a control cell can be compared in the presence of the test agent.
  • the control cell is substantially similar to the cell that has the one or more proteins but does not contain the one or more proteins.
  • Exemplary proteins include those that are at least about 52% homologous to a protein encoded by a Drosophila gene over at least about 54% of the encoded protein.
  • the Drosophila gene is one that, when altered in its expression, results in altered intracellular calcium in a Drosophila cell, altered store-operated calcium entry into a Drosophila cell and/or altered basal or resting cytosolic calcium level in a Drosophila cell.
  • the Drosophila gene is CG8743.
  • the one or more proteins that is (are) at least about 52% homologous to a protein encoded by a Drosophila gene over at least about 54% of the encoded protein does not contain the sequence of amino acids EWKFAR.
  • At least one of the one or more proteins that is (are) at least about 52% homologous to a protein encoded by a Drosophila gene over at least about 54% of the encoded protein is an ion transport protein or a calcium-binding protein.
  • Other exemplary proteins include mucolipidin, mucolipidin-like proteins, Homo sapiens mucolipin-3, Mus musculus mucolipin-3, C. efegans CUP-5 protein, Homo sapiens mucolipin 1 , Mus musculus mucolipin-2, Mus musculus mucolipin 1 and Homo sapiens mucolipidosis type IV protein. Additional proteins suitable for use in the methods include those having an amino acid sequence selected from the following: GenBank accession No.
  • GenBank accession No. AAL84622 SEQ ID NO: 4, GenBank accession No. AAL84623, SEQ ID NO: 6, Genbank accession No. BAB29372, SEQ ID NO: 8, GenBank accession No. AAL38964, SEQ ID NO: 10, GenBank accession No. NP 498664, SEQ ID NO: 12, GenBank accession No. NP_498665, SEQ ID NO: 14, GenBank accession No. CAC0821 5, SEQ ID NO: 1 6, GenBank accession No. AAL89754, SEQ ID NO: 18, GenBank accession No. AAK19624, SEQ ID NO: 20, GenBank accession No. AAL38965, SEQ ID NO: 22, GenBank accession No. AAH05651 , SEQ ID NO: 24, GenBank accession No.
  • AAG42242 SEQ ID NO: 26, GenBank accession No. BAA91951 , SEQ ID NO: 28, GenBank accession No. CAC07813, SEQ ID NO: 30, GenBank accession No. XP 088847, SEQ ID NO: 32, Genbank accession No. NP_598921 , SEQ ID NO: 34, GenBank accession No. AAM1 5598, SEQ ID NO: 36, Genbank accession No. AAM1 5597, SEQ ID NO: 38, Genbank accession No. AAM28596, SEQ ID NO: 40, Genbank accession No. NP 080932, SEQ ID NO: 42, GenBank accession No. NP 065394, SEQ ID NO 44, GenBank accession No.
  • NP 444407 SEQ ID NO: 46, GenBank accession No. NP_060768, SEQ ID NO: 48, GenBank accession No. CAC0781 3, SEQ ID NO: 50, GenBank accession No. AAM1 5596, SEQ ID NO: 52, GenBank accession No. AAL58667, SEQ ID NO: 54, GenBank accession No. AAH05149, SEQ ID NO: 56, GenBank accession No. AAG00798, SEQ ID NO: 58, GenBank accession No. AAG00797, SEQ ID NO: 60, GenBank accession No. BAB31730, SEQ ID NO: 62, GenBank accession No.
  • AAG10422 and SEQ ID NO: 64 Calcium levels can be evaluated in a variety of ways.
  • store-operated calcium entry into the cell is evaluated and compared.
  • a test agent is identified as an agent that modulates calcium levels if store-operated calcium entry into the cell differs in the presence and absence of the test agent or if store-operated calcium entry into the cell and the control cell differs. In the absence of the test agent the cell will exhibits store-operated calcium entry.
  • intracellular or cytoplasmic calcium levels in the cell are evaluated and compared in the presence and absence of the test agent.
  • intracellular or cytoplasmic calcium levels of the cell and a control cell in the presence of the test agent are evaluated and compared.
  • a test agent is identified as an agent that modulates intracellular calcium if the intracellular or cytoplasmic calcium levels of the cell differ in the presence and absence of the test agent or if the intracellular or cytoplasmic calcium levels of the cell and the control cell differ.
  • calcium levels may be evaluated by monitoring intracellular calcium levels over time for fluctuations in intracellular calcium levels.
  • the intracellular calcium level time courses of the cell are then compared in the presence and absence of the test agent or the intracellular calcium level time courses of the cell and a control cell are compared in the presence of the test agent.
  • a test agent is identified as an agent that modulates intracellular calcium if the intracellular calcium level time courses of the cell differ in the presence and absence of the test agent or if the intracellular calcium level time courses of the cell and the control cell differ.
  • calcium levels may be evaluated by assessing ion flux across the cell plasma membrane.
  • the ion flux across the cell plasma membrane is then compared in the presence and absence of the test agent or the ion flux across plasma membrane of the cell and a control cell is compared in the presence of the test agent.
  • a test agent is identified as an agent that modulates intracellular calcium if the ion flux across the cell plasma membrane differs in the presence and absence of the test agent or if the ion flux across the plasma membrane of the cell and the control cell differs.
  • a step of reducing the calcium level in an intracellular calcium store may be performed before, simultaneous with or after the step of contacting the cell with the test agent.
  • calcium enters the cell upon reduction of calcium levels in intracellular calcium stores in the absence of the test agent.
  • methods of modulating intracellular calcium in a cell include modulating the level and/or activity of one or more proteins.
  • Exemplary proteins whose level and/or activity may be modulated include those that are at least about 52% homologous to a protein encoded by a Drosophila gene over at least about 54% of the encoded protein.
  • the Drosophila gene is one that, when altered in its expression, results in altered intracellular calcium in a Drosophila cell, altered store-operated calcium entry into a Drosophila cell and/or altered basal or resting cytosolic calcium level in a Drosophila cell.
  • the Drosophila gene is CG8743.
  • the one or more proteins that is (are) at least about 52% homologous to a protein encoded by a Drosophila gene over at least about 54% of the encoded protein does not contain the sequence of amino acids EWKFAR.
  • at least one of the one or more proteins that is (are) at least about 52% homologous to a protein encoded by a Drosophila gene over at least about 54% of the encoded protein is an ion transport protein or a calcium- binding protein.
  • exemplary proteins include mucolipidin, mucolipidin-like proteins, Homo sapiens mucolipin-3, Mus musculus mucolipin-3, C. elegans CUP-5 protein, Homo sapiens mucolipin 1 , Mus musculus mucolipin-2, Mus musculus mucolipin 1 and Homo sapiens mucolipidosis type IV protein.
  • Additional proteins suitable for use in the methods include those having an amino acid sequence selected from the following: GenBank accession No. AAL84622, SEQ ID NO: 4, GenBank accession No. AAL84623, SEQ ID NO: 6, Genbank accession No. BAB29372, SEQ ID NO: 8, GenBank accession No. AAL38964, SEQ ID NO: 10, GenBank accession No.
  • NP_498664 SEQ ID NO: 1 2, GenBank accession No. NP_498665, SEQ ID NO: 14, GenBank accession No. CAC0821 5, SEQ ID NO: 1 6, GenBank accession No. AAL89754, SEQ ID NO: 1 8, GenBank accession No. AAK1 9624, SEQ ID NO: 20, GenBank accession No. AAL38965, SEQ ID NO: 22, GenBank accession No. AAH05651 , SEQ ID NO: 24, GenBank accession No. AAG42242, SEQ ID NO: 26, GenBank accession No. BAA91 951 , SEQ ID NO: 28, GenBank accession No. CAC0781 3, SEQ ID NO: 30, GenBank accession No.
  • GenBank accession No. NP_598921 GenBank accession No. AAM1 5598, SEQ ID NO: 36, Genbank accession No. AAM15597, SEQ ID NO: 38, Genbank accession No. AAM28596, SEQ ID NO: 40, Genbank accession No. NP 080932, SEQ ID NO: 42, GenBank accession No. NP 065394, SEQ ID NO 44, GenBank accession No. NP 444407, SEQ ID NO: 46, GenBank accession No. NP_060768, SEQ ID NO: 48, GenBank accession No. CAC07813, SEQ ID NO: 50, GenBank accession No.
  • Candidate agents for use in treating or preventing a disease or disorder may be identified by evaluating and comparing calcium levels in a test cell having a polymorphic form of one or more proteins involved in calcium homeostasis in the presence and absence of a test agent.
  • the polymorphic form of the one or more proteins is a form that is not a wild-type form of the protein.
  • calcium levels in the test cell in the presence of the test agent and in a control cell can be compared.
  • the control cell contains a wild-type form of the one or more proteins and calcium levels in the test cell differ from calcium levels in the control cell.
  • a test agent is identified as a candidate agent for use in treating a disease or disorder if calcium levels in the test cell differ in the presence and absence of the test agent or if calcium levels in the test cell in the presence of the test agent are similar to calcium levels in the control cell.
  • Candidate agents for use in treating or preventing a disease or disorder may also be identified by evaluating and comparing calcium levels in a non- human transgenic animal having a polymorphic form of one or more proteins involved in calcium homeostasis in the presence and absence of a test agent.
  • the polymorphic form of the one or more proteins is a form that is not a wild- type form of the protein.
  • calcium levels in the non-human transgenic animal in the presence of the test agent and in a control animal can be compared.
  • the control animal contains a wild-type form of the one or more proteins and calcium levels in the transgenic animal differ from calcium levels in the control animal.
  • a test agent is identified as a candidate agent for use in treating a disease or disorder if calcium levels in the transgenic animal differ in the presence and absence of the test agent, if calcium levels in the transgenic animal in the presence of the test agent are similar to calcium levels in the control animal, or if in the presence of the test agent the transgenic animal exhibits a phenotype similar to that of the control animal.
  • Exemplary proteins include any polymorphic form of a protein that is at least about 52% homologous to a protein encoded by a Drosophila gene over at least about 54% of the encoded protein.
  • the Drosophila gene is one that, when altered in its expression, results in altered intracellular calcium in a Drosophila cell, altered store-operated calcium entry into a Drosophila cell and/or altered basal or resting cytosolic calcium level in a Drosophila cell.
  • the Drosophila gene is CG8743.
  • the one or more proteins that is (are) at least about 52% homologous to a protein encoded by a Drosophila gene over at least about 54% of the encoded protein does not contain the sequence of amino acids EWKFAR.
  • at least one of the one or more proteins that is (are) at least about 52% homologous to a protein encoded by a Drosophila gene over at least about 54% of the encoded protein is an ion transport protein or a calcium-binding protein.
  • Other exemplary proteins include any polymorphic form of mucolipidin, mucolipidin-like proteins, Homo sapiens mucolipin-3, Mus musculus mucolipin-3, C.
  • elegans CUP-5 protein Homo sapiens mucolipin 1 , Mus musculus mucolipin- 2, Mus musculus mucolipin 1 and Homo sapiens mucolipidosis type IV protein.
  • Additional proteins suitable for use in the methods include those having an amino acid sequence selected from the following: GenBank accession No.
  • GenBank accession No. AAL84622 SEQ ID NO: 4, GenBank accession No. AAL84623, SEQ ID NO: 6, Genbank accession No. BAB29372, SEQ ID NO: 8, GenBank accession No. AAL38964, SEQ ID NO: 10, GenBank accession No. NP_498664, SEQ ID NO: 1 2, GenBank accession No. NP_498665, SEQ ID NO: 14, GenBank accession No. CAC0821 5, SEQ ID NO: 1 6, GenBank accession No. AAL89754, SEQ ID NO: 18, GenBank accession No. AAK19624, SEQ ID NO: 20, GenBank accession No. AAL38965, SEQ ID NO: 22, GenBank accession No.
  • GenBank accession No. AAH05651 SEQ ID NO: 24, GenBank accession No. AAG42242, SEQ ID NO: 26, GenBank accession No. BAA91951 , SEQ ID NO: 28, GenBank accession No. CAC07813, SEQ ID NO: 30, GenBank accession No. XP 088847, SEQ ID NO: 32, Genbank accession No. NP 598921 , SEQ ID NO: 34, GenBank accession No. AAM1 5598, SEQ ID NO: 36, Genbank accession No. AAM1 5597, SEQ ID NO: 38, Genbank accession No. AAM28596, SEQ ID NO: 40, Genbank accession No. NP 080932, SEQ ID NO: 42, GenBank accession No.
  • NP_065394 SEQ ID NO 44, GenBank accession No. NP 444407, SEQ ID NO: 46, GenBank accession No. NP 060768, SEQ ID NO: 48, GenBank accession No. CAC07813, SEQ ID NO: 50, GenBank accession No. AAM1 5596, SEQ ID NO: 52, GenBank accession No. AAL58667, SEQ ID NO: 54, GenBank accession No. AAH05149, SEQ ID NO: 56, GenBank accession No. AAG00798, SEQ ID NO: 58, GenBank accession No. AAG00797, SEQ ID NO: 60, GenBank accession No. BAB31730, SEQ ID NO: 62, GenBank accession No.
  • AAG10422 and SEQ ID NO: 64 The calcium levels that may be evaluated and compared as described in the above methods include store-operated calcium entry and intracellular and/or cytoplasmic levels. In addition, intracellular calcium levels may be monitored over time for fluctuations in intracellular calcium levels and/or ion flux across the plasma membrane may be assessed. In a particular embodiment, candidate agents are identified for use in the treatment of a disease or disorder. Exemplary diseases and disorders include, but are not limited to, endocytosis, membrane trafficking, and lysosomal storage diseases and disorders, such as Mucolipidosis Type IV, and/or calcium dyshomeostasis or altered intracellular calcium regulation.
  • Candidate agents are identified by contacting a cell or organism expressing a phenotype of the disease or disorder with a test agent that modulates cellular calcium levels. Such levels may include, for example, store-operated calcium entry and/or basal, resting cytosolic calcium levels.
  • the symptoms of the disease or disorder are evaluated and compared in the cell or organism in the presence and absence of the test agent or symptoms of the disease or disorder are evaluated and compared in the cell or organism in the presence of the test agent and a wild- type cell or organism.
  • a test agent is identified as a candidate agent for use in treatment of the disease or disorder if symptoms of the disease or disorder in the cell or organism are ameliorated in the presence of the test agent or if the cell or organism exhibits at least partial restoration of the wild-type phenotype in the presence of the agent. In the absence of the disease or disorder the cell or at least some cells of the organism exhibit store-operated calcium entry. The wild- type cell or organism does not express the disease phenotype.
  • the agent may modulate store-operated calcium entry and/or basal/resting cytosolic calcium levels.
  • the disease or disorder may include endocytosis, membrane trafficking, and lysosomal storage diseases and disorders, such as Mucolipidosis Type IV, and/or calcium dyshomeostasis or altered intracellular calcium regulation.
  • the disease or disorder may be the result of an altered gene.
  • the gene may encode a protein involved in calcium homeostasis including modulation of intracellular calcium levels, store-operated calcium entry, and/or basal/resting cytosolic calcium levels.
  • the encoded protein can be a protein that is at least about 52% homologous to a protein encoded by a Drosophila gene over at least about 54% of the encoded protein.
  • the Drosophila gene is one that, when altered in its expression, results in altered intracellular calcium in a Drosophila cell, altered store-operated calcium entry into a Drosophila cell and/or altered basal or resting cytosolic calcium level in a Drosophila cell.
  • the Drosophila gene is CG8743.
  • the one or more proteins that is (are) at least about 52% homologous to a protein encoded by a Drosophila gene over at least about 54% of the encoded protein does not contain the sequence of amino acids EWKFAR.
  • at least one of the one or more proteins that is (are) at least about 52% homologous to a protein encoded by a Drosophila gene over at least about 54% of the encoded protein is an ion transport protein or a calcium-binding protein.
  • the encoded protein may also be a mucolipidin, mucolipidin-like protein, Homo sapiens mucolipin-3, Mus musculus mucolipin-3, C. elegans CUP-5 protein, Homo sapiens mucolipin 1 , Mus musculus mucolipin-2, Mus musculus mucolipin 1 and Homo sapiens mucolipidosis type IV protein.
  • Additional proteins suitable for use in the methods include those having an amino acid sequence selected from the following: GenBank accession No. AAL84622, SEQ ID NO: 4, GenBank accession No. AAL84623, SEQ ID NO: 6, Genbank accession No.
  • GenBank accession No. AAL38964 SEQ ID NO: 10, GenBank accession No. NP 498664, SEQ ID NO: 1 2, GenBank accession No. NP 498665, SEQ ID NO: 14, GenBank accession No. CAC0821 5, SEQ ID NO: 1 6, GenBank accession No. AAL89754, SEQ ID NO: 1 8, GenBank accession No. AAK1 9624, SEQ ID NO: 20, GenBank accession No. AAL38965, SEQ ID NO: 22, GenBank accession No. AAH05651 , SEQ ID NO: 24, GenBank accession No. AAG42242, SEQ ID NO: 26, GenBank accession No.
  • NP_060768 SEQ ID NO: 48, GenBank accession No. CAC07813, SEQ ID NO: 50, GenBank accession No. AAM1 5596, SEQ ID NO: 52, GenBank accession No. AAL58667, SEQ ID NO: 54, GenBank accession No. AAH05149, SEQ ID NO: 56, GenBank accession No. AAG00798, SEQ ID NO: 58, GenBank accession No. AAG00797, SEQ ID NO: 60, GenBank accession No. BAB31730, SEQ ID NO: 62, GenBank accession No.
  • AAG10422 and SEQ ID NO: 64. that is at least about 52% homologous to a protein encoded by a Drosophila gene over at least about 54% of the encoded protein. Also provided are methods of modulating lipid transport and/or storage in a cell by altering intracellular calcium in a cell, cytoplasmic calcium in a cell, and/or store-operated calcium entry into the cell.
  • the cells used in the methods can be mammalian cells such as, for example, rodent or human cells.
  • the cell may be a recombinant cell and at least one of the one or more proteins may be heterologous to the cell.
  • calcium homeostasis refers to the maintenance of an overall balance in intracellular calcium levels and movements, including calcium signalling, within a cell.
  • calcium dyshomeostasis refers to altered, abnormal or impaired calcium homeostasis.
  • calcium dyshomeostasis can be imbalances or disturbances in intracellular calcium levels or movements such as may result from altered calcium regulation in a cell.
  • intracellular calcium refers to calcium located in a cell without specification of a particular cellular location.
  • cytosolic or “cytoplasmic” with reference to calcium refers to calcium located in the cell cytoplasm.
  • modulation with reference to intracellular calcium refers to any alteration or adjustment in intracellular calcium including but not limited to alteration of calcium concentration in the cytoplasm and/or intracellular calcium storage organelles, e.g., endoplasmic reticulum, and alteration of the kinetics of calcium fluxes into, out of and within cells.
  • basal or resting with reference to cytosolic calcium levels refers to the concentration of calcium in the cytoplasm of a cell, such as, for example, an unstimulated cell, that has not been subjected to a condition that results in movement of calcium into or out of the cell or within the cell.
  • the basal or resting cytosolic calcium level can be the concentration of free calcium (i.e., calcium that is not bound to a cellular calcium-binding substance) in the cytoplasm of a cell, such as, for example, an unstimulated cell, that has not been subjected to a condition that results in movement of calcium into or out of the cell.
  • movement with respect to ions, including cations, e.g., calcium, refers to movement or relocation, such as for example flux, of ions into, out of, or within a cell.
  • movement of ions can be, for example, movement of ions from the extracellular medium into a cell, from within a cell to the extracellular medium, from within an intracellular organelle or storage site to the cytosol, from the cytosol into an intracellular organelle or storage site, from one intracellular organelle or storage site to another intracellular organelle or storage site, from the extracellular medium into an intracellular organelle or storage site, from an intracellular organelle or storage site to the extracellular medium and from one location to another within the cell cytoplasm.
  • cation entry or “calcium entry” into a cell refers to entry of cations, such as calcium, into an intracellular location, such as the cytoplasm of a cell or into the lumen of an intracellular organelle or storage site.
  • cation entry can be, for example, the movement of cations into the cell cytoplasm from the extracellular medium or from an intracellular organelle or storage site, or the movement of cations into an intracellular organelle or storage site from the cytoplasm or extracellular medium. Movement of calcium into the cytoplasm from an intracellular organelle or storage site is also referred to as "calcium release" from the organelle or storage site.
  • protein that modulates intracellular calcium refers to any cellular protein that is involved in regulating, controlling and/or altering intracellular calcium.
  • a protein can be involved in altering or adjusting intracellular calcium in a number of ways, including, but not limited to, through the maintenance of resting or basal cytoplasmic calcium levels, or through involvement in a cellular response to a signal that is transmitted in a cell through a mechanism that includes a deviation in intracellular calcium from resting or basal states.
  • a "cellular" protein is one that is associated with a cell, such as, for example, a cytoplasmic protein, a plasma membrane-associated protein or an intracellular membrane protein.
  • Proteins that modulate intracellular calcium include, but are not limited to, ion transport proteins, calcium-binding proteins and regulatory proteins that regulate ion transport proteins.
  • agent that modulates intracellular calcium refers to any substance that can modulate intracellular calcium.
  • agents include, but are not limited to, small organic molecules, large organic molecules, amino acids, peptides, polypeptides, nucleotides, nucleic acids (including DNA, cDNA, RNA, antisense RNA and any double- or single-stranded forms of nucleic acids), polynucleotides, carbohydrates, lipids, lipoproteins, glycoproteins, inorganic ions (including, for example, Gd 3 + lead and lanthinum).
  • test agent in the context of methods for identifying agents that modulate intracellular calcium, refers to any substance that is being evaluated as a possible agent that modulates intracellular calcium.
  • agent that modulates the level and/or activity of a protein refers to any substance that can modulate the amount of and/or activity of a protein.
  • agents include, but are not limited to, small and large organic molecules, amino acids, peptides, polypeptides, nucleotides, nucleic acids (including DNA, cDNA, expression vectors, RNA, antisense RNA, and any double- or single-stranded forms of nucleic acids), polynucleotides, carbohydrates, lipids, lipoproteins and glycoproteins.
  • amelioration refers to an improvement in a disease or condition or at least a partial relief of symptoms associated with a disease or condition.
  • cell response refers to any cellular response that results from ion movement into or out of a cell or within a cell.
  • the cell response may be associated with any cellular activity that is dependent, at least in part, on ions such as, for example, calcium.
  • Such activities may include, for example, cellular activation, gene expression, endocytosis, exocytosis, cellular trafficking and apoptotic cell death.
  • heterologous nucleic acids As used herein, “heterologous” or “foreign” with reference to nucleic acids, cDNA, DNA and RNA are used interchangeably and refer to nucleic acid, DNA or RNA that does not occur naturally as part of the genome in which it is present or which is found in a location(s) or in an amount in the genome that differs from that in which it occurs in nature. It is nucleic acid that has been exogenously introduced into the cell. Thus, heterologous nucleic acid is nucleic acid not normally found in the host genome in an identical context. Examples of heterologous nucleic acids include, but are not limited to, DNA that encodes a gene product or gene product(s) of interest, introduced, for example, for purposes of gene therapy or for production of an encoded protein. Other examples of heterologous DNA include, but are not limited to, DNA that encodes a selectable marker, DNA that encodes therapeutically effective substances, such as anti-cancer agents, enzymes and hormones, and DNA that encodes other types of
  • expression refers to the process by which nucleic acid, e.g., DNA, is transcribed into mRNA and translated into peptides, polypeptides, or proteins. If the nucleic acid is derived from genomic DNA, expression may, if an appropriate eukaryotic host cell or organism is selected, include splicing of the mRNA.
  • vector or "plasmid” refers to discrete elements that are used to introduce heterologous nucleic acids into cells. Typically, vectors are used to transfer heterologous nucleic acids into cells for either expression of the heterologous nucleic acid or for replication of the heterologous nucleic acid. Selection and use of such vectors and plasmids are well within the level of skill of the art.
  • transfection refers to the process by which nucleic acids are introduced into cells. Transfection refers to the taking up of exogenous nucleic acid, e.g., an expression vector, by a host cell whether or not any coding sequences are in fact expressed. Numerous methods of transfection are known to the ordinarily skilled artisan. Successful transfection is generally recognized by detection of the presence of the heterologous nucleic acid within the transfected cell, such as, for example, any visualization of the heterologous nucleic acid or any indication of the operation of a vector within the host cell.
  • injection refers to the microinjection (use of a small syringe) of nucleic acid into a cell.
  • amino acids which occur in the various amino acid sequences appearing herein, are identified according to their well-known, three- letter or one-letter abbreviations (see Table 1 ). The nucleotides, which occur in the various DNA fragments, are designated with the standard single-letter designations used routinely in the art.
  • amino acid residue refers to an amino acid formed upon chemical digestion (hydrolysis) of a polypeptide at its peptide linkages. The amino acid residues described herein are preferably in the " " isomeric form.
  • NH 2 refers to the free amino group present at the amino terminus of a polypeptide.
  • COOH refers to the free carboxy group present at the carboxyl terminus of a polypeptide.
  • amino acid residue sequences represented herein by formulae have a left to right orientation in the conventional direction of amino-terminus to carboxyl-terminus.
  • amino acid residue is broadly defined to include the amino acids listed in the Table of Correspondence and modified and unusual amino acids, such as those referred to in 37 C.F.R. ⁇ ⁇ 1 .821 -1 .822, and incorporated herein by reference.
  • a dash at the beginning or end of an amino acid residue sequence indicates a peptide bond to a further sequence of one or more amino acid residues or to an amino-terminal group such as NH 2 or to a carboxyl-terminal group such as COOH.
  • similarity between two proteins or nucleic acids refers to the relatedness between the amino acid sequences of the proteins or the nucleotide sequences of the nucleic acids. Similarity can be based on the degree of identity and/or homology of sequences and the residues contained therein. Methods for assessing the degree of similarity between proteins or nucleic acids are known to those of skill in the art. For example, in one method of assessing sequence similarity, two amino acid or nucleotide sequences are aligned in a manner that yields a maximal level of identity between the sequences. "Identity” refers to the extent to which the amino acid or nucleotide sequences are invariant.
  • Alignment of amino acid sequences, and to some extent nucleotide sequences, also can take into account conservative differences and/or frequent substitutions in amino acids (or nucleotides). Conservative differences are those that preserve the physico-chemical properties of the residues involved. Alignments can be global (alignment of the compared sequences over the entire length of the sequences and including all residues) or local (the alignment of a portion of the sequences that includes only the most similar region or regions). "Identity" per se has an art-recognized meaning and can be calculated using published techniques. (See, e.g.
  • identity is well known to skilled artisans (Carillo, H. & Lipton, D., SIAM J Applied Math 48: 1073 (1 988)).
  • homology with reference to proteins or nucleic acids refers to shared sequence similarity that takes into account both identical residues and residues that may substitute for one another. Substitutions may include, for example, conserved amino acids and frequent substitutions based on statistical analyses and evolutionary distance.
  • Percent identity and percent homology may be determined, for example, by comparing sequence information using any of a number of computer algorithms known in the art.
  • the GAP program uses the alignment method of Needleman and Wunsch (J. Mol. Biol. 48:443 (1970)), as revised by Smith and Waterman [Adv. Appl. Math. 2:482 (1981 )). Briefly, the GAP program defines similarity as the number of aligned symbols (i.e., nucleotides or amino acids) which are similar, divided by the total number of symbols in the shorter of the two sequences.
  • Default parameters for the GAP program may include: (1 ) a unary comparison matrix (containing a value of 1 for identities and 0 for non-identities) and the weighted comparison matrix of Gribskov and Burgess, Nucl. Acids Res. 14:6745 (1 986), as described by Schwartz and Dayhoff, eds., A TLAS OF PROTEIN SEQUENCE AND STRUCTURE, National Biomedical
  • identity can be determined using known computer algorithms such as the "FAST A” program, using for example, the default parameters as in Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85:2444 (1 988).
  • the BLAST function of the National Center for Biotechnology Information database may be used to determine identity.
  • a level of 90% or more identity between the polypeptides is indicative of having no more than 10% (i.e., 10 out of 100) amino acids in the test polypeptide differing from that of the reference polypeptide.
  • Similar comparisons may be made between a test and reference polynucleotides. Such differences may be represented as point mutations randomly distributed over the entire length of an amino acid sequence or they may be clustered in one or more locations of varying length up to the maximum allowable, e.g. 10/100 amino acid difference (approximately 90% identity).
  • the BLinK tool (“BLAST Link”) displays the results of precomputed BLAST searches (Altschul et al. (1 9990) J. Mol. Biol. 275:403-410) that have been done for protein sequences in the Entrez Proteins data domain against the non-redundant (nr) database (e.g., GenBank).
  • Methods commonly employed to determine identity or homology between two sequences include, but are not limited to, those disclosed in Guide to Huge Computers, Martin J. Bishop, ed., Academic Press, San Diego, 1 994, and Carillo, H. & Lipton, D., SIAM J Applied Math 43:1073 (1988). Methods to determine identity and homology are codified in computer programs.
  • Computer program methods to determine identity and homology between two sequences include, but are not limited to, GCG program package (Devereux, J., et al., Nucleic Acids Research 12(0:387 (1984)), BLASTP, BLASTN, FASTA (Atschul, S.F., et a/., J Molec Biol 215:403 (1 990)). Therefore, as used herein, the terms "identity” and "homology” represent a comparison between two polypeptides or polynucleotides.
  • a "test" polypeptide or polynucleotide is compared to a "reference” molecule to determine if there is a significant level of similarity (including identity and/or homology) between the test and reference molecules.
  • the terms at least "X% homology over X% of the protein” or "X% identity over X% of the protein” with reference to a comparison of protein A with protein B refers to protein A having X% homology or X% identity to protein B over X% of the amino acid sequence of protein B.
  • the term “over the protein” means over the length of the amino acid sequence of the protein, but not necessarily over a contiguous sequence of amino acids of the protein.
  • protein B may be 40% homologous to protein A over 60% of protein B; however, the 60% of the amino acids in protein B to which protein A has homology may be located in multiple, separate sequences (e.g., regions or domains) of protein B.
  • protein "homologs” refers to similar proteins encoded by related but different genes either within a species or between species. Protein “orthologs” refers to similar proteins in different species that arose from a common ancestral gene.
  • all assays and procedures, such as hybridization reactions and antibody-antigen reactions, unless otherwise specified, are conducted under conditions recognized by those of skill in the art as standard conditions.
  • Cellular calcium homeostasis is a result of the summation of regulatory systems involved in the control of intracellular calcium levels and movements.
  • Cellular calcium homeostasis is achieved, at least in part, by calcium binding and by movement of calcium into and out of the cell across the plasma membrane and within the cell by movement of calcium across membranes of intracellular organelles including, for example, the endoplasmic reticulum, sarcoplasmic reticulum, mitochondria and endocytic organelles including endosomes and lysosomes.
  • Fluctuations in the level of calcium ions in cells provide important biological signals involved in processes such as protein secretion, muscle contraction, cell death and development.
  • the movement of cations, such as calcium, into, within and out of cells thus plays a critical role in the operation and survival of cells.
  • Calcium movement into, out of and within cells can act as a signal which is highly organized in space, frequency and amplitude due in part to localization of the movements and tight regulation of the processes through which calcium movement occurs in cells.
  • Alterations in the elements involved in movement of calcium ions, and/or in the regulation thereof, can lead to a plethora of diseases. Altered calcium regulation or calcium dyshomeostasis in cells is associated with many diseases and disorders.
  • cellular proteins that modulate intracellular calcium including, for example, proteins that provide for and/or regulate the movement of cations, such as calcium, into, within and out of cells, and to identify agents that modulate intracellular calcium.
  • Methods are provided herein for screening for and identifying candidate cellular proteins involved in modulating intracellular calcium.
  • the methods are for screening for and identifying candidate ion transport proteins that provide for movement of cations, such as calcium, into, out of and within cells and proteins that regulate such ion transport proteins.
  • methods for identifying agents that modulate intracellular calcium and methods for modulating intracellular calcium and for treating diseases and disorders are also provided herein.
  • Calcium from the extracellular space can enter the cell through various calcium channels and a sodium/calcium exchanger and is actively extruded from the cell by calcium pumps and sodium/calcium exchangers.
  • Calcium can also be released from internal stores through inositol triphosphate or ryanodine receptors and can be taken up by these organelles by means of calcium pumps.
  • Endocytosis provides another process by which cells can take up calcium from the extracellular medium through endosomes.
  • some cells e.g., exocrine cells, can release calcium via exocytosis.
  • Methods provided herein for identifying cellular proteins involved in modulating intracellular calcium can be used to identify proteins that participate in any of a number of processes that affect intracellular calcium, including, but not limited to, receptor-mediated and second messenger-operated calcium movement, calcium uptake and release by endosomes and lysosomes and store- operated calcium entry into cells.
  • Cytosolic calcium concentration is tightly regulated with resting levels usually estimated at approximately 0.1 ⁇ M in mammalian cells, whereas the extracellular calcium concentration is typically about 2 mM. This tight regulation facilitates transduction of signals into and within cells through transient calcium flux across the plasma membrane and membranes of intracellular organelles. As a result, disturbance of resting cytosolic calcium levels can effect transmission of such signals and give rise to defects in a myriad number of cellular processes. For example, cell proliferation involves a prolonged calcium signalling sequence. Other cellular processes, including but not limited to, secretion, fertilization and learning, involve calcium signalling. 2. Receptor-mediated and second messenger-operated cation movement
  • Receptor-mediated cation channels are gated in response to ligand binding to a membrane receptor distinct from the channel protein itself. Some receptor- mediated cation channels are activated downstream of tyrosine kinases and others via G protein signaling cascades. Receptor-mediated channels are expressed in a number of both excitable and nonexcitable cells, including smooth muscle, mast cells, epidermis and renal mesangial cells.
  • receptor-mediated cation channels are regulated through second messengers induced in response to ligand-binding to a membrane receptor.
  • Such cation channels are referred to as second messenger- operated channels.
  • cyclic nucleotides generated by adenylyl and guanylyl cyclases can directly activate cation-permeable channels.
  • Such cyclic nucleotide-gated channels are predominantly expressed in sensory tissues, for example the retina and in olfactory/gustatory epithelia.
  • Calcium is another second messenger that can mediate ion channel function. Examples of calcium- mediated channels include calcium-activated potassium and chloride channels as well as cation channels in neutrophils, smooth muscle and mast cells.
  • Inositol phosphates generated upon activation of phospholipase C can also act as second messengers that activate certain channels.
  • channels responsive to inositol-1 ,4,5-triphosphate (IP 3 ) include the intracellular IP 3 receptor of the endoplasmic reticulum as well as plasma membrane channels such as those expressed in T-lymphocytes, mast cells and epidermal cells.
  • the intracellular IP 3 receptor functions as a ligand-gated ion channel that permits passage of calcium upon binding of IP 3 released through hydolysis of membrane phospholipids by activated phospholipase C (PLC).
  • PLC can be activated through agonist binding to a surface membrane G protein-coupled receptor.
  • IP 3 Activation of the IP 3 receptor results in the release of calcium stored in the endoplasmic reticulum into the cytoplasm which produces a transient "peak" increase in cytosolic calcium concentration.
  • IP 3 -dependent agonists appear to be associated with calcium release from lysosomes in MDCK cells (see Haller et al. (1 996) Biochem. J. 3 /3:909-91 2).
  • Lipids and polyunsaturated fatty acids may also act as second messengers for the activation of ion channels.
  • PUFAs polyunsaturated fatty acids
  • arachidonic acid and its metabolites, as well as linolenic acid can activate receptor-mediated ion channels such as Drosophila TRP and TRPL channels (see, e.g., Chyb et al. (1 999) Nature 337:255-259).
  • phospholipids such as lysophospholipids (e._/.,lysophosphatidic acid (LPA), lysophosphatidylcholine (LPC), sphingosylphosphoryl choline (SPC) and sphingosine 1 -phosphate (SIP))
  • LPA lysophosphatidic acid
  • LPC lysophosphatidylcholine
  • SPC sphingosylphosphoryl choline
  • SIP sphingosine 1 -phosphate
  • GPCRs G-protein-coupled receptors
  • PLC generates not only IP 3 but also diacylglycerol (DAG) which is a potential precursor for polyunsaturated fatty acids.
  • DAG diacylglycerol
  • Endocytosis is a process whereby contents of the cell plasma membrane and extracellular medium are transported into the interior of the cell. Not only does endocytosis serve "house-keeping" functions of a cell, it plays crucial roles in cell signaling, development, and the regulation of varied biological processes including, for example, synaptogenesis, neural plasticity, generation of morphogen gradients and programmed cell death. Endocytosis may have both negative and positive influences on signaling. For example, endocytosis can regulate the number of receptors on the plasma membrane. In addition, endocytosis plays a positive role in signaling mediated by the Notch signaling pathway which acts to determine cell-type specificity during development.
  • Endosomes are morphologically heterogeneous and constitute a pleiomorphic smooth membrane system of tubular and vesicular elements.
  • the vesicular elements contain intra-organelle vesicles and are described as multivesicular bodies.
  • Endocytosed macromolecules are delivered first to early endosomes and then to late endosomes.
  • Early endosomes are tubular with varicosities and many are located peripherally within the cell close to the plasma membrane. Late endosomes are more spherical and have the appearance of multivesicular bodies.
  • Lysosomes are membrane-bound organelles containing hydrolytic enzymes and are regarded as the terminal degradation compartment of the endocytic pathway. Lysosomes also play an important role in phagocytosis, autophagy, crinophagy and proteolysis of some cytosolic proteins that are transported across the lysosomal membrane.
  • lysosomes secrete their contents after fusion with the plasma membrane.
  • the limiting membrane of lysosomes contains a set of highly glycosylated lysosomal- associated membrane proteins (LAMPs). Additional lysosomal membrane proteins mediate transport of ions, amino acids, and other solutes across the lysosomal membrane.
  • LAMPs highly glycosylated lysosomal- associated membrane proteins
  • Additional lysosomal membrane proteins mediate transport of ions, amino acids, and other solutes across the lysosomal membrane.
  • the organelles of the late endocytic pathway interact with each other and are in dynamic equilibrium. Content mixing and/or exchange of membrane proteins occurs between late endosomes, lysosomes and between late endososomes and lysosomes.
  • endocytosed macromolecules to lysosomes occurs by content mixing between late endosomes and lysosomes as a result of transient as well as direct fusion which can form hybrid organelles.
  • Cell-free heterotypic fusion of mammalian late endosomes and lysosomes requires calcium which may mediate its effects via calmodulin.
  • the calcium is derived and released from the endocytic organelle lumen and is required in a late step in fusion. Although the calcium release pathway has not yet been elucidated, docking of endocytic organelles is thought to trigger release of endocytosed calcium from the lumen of endocytic organelles into the cytoplasm.
  • Lysosomes contain a mobilizable calcium pool. Furthermore, although no cation transport molecule has been identified in endosomes or lysosomes, both endosomal and lysosomal membranes provide for calcium transport. Thus, uptake and release of calcium from endosomes and lysosomes can impact intracellular and cytosolic calcium levels.
  • CCE capacitative calcium entry
  • Store-operated calcium entry can be recorded as an ionic current with some characteristic properties. In some instances, this current is referred to as l soc (store-operated current) or l CRAC (calcium release- activated current).
  • Intracellular calcium stores can be characterized by sensitivity to agents, which can be physiological or pharmacological, that activate release of calcium from the stores or inhibit uptake of calcium into the stores.
  • agents which can be physiological or pharmacological, that activate release of calcium from the stores or inhibit uptake of calcium into the stores.
  • Different cells have been studied in characterization of intracellular calcium stores, and stores have been characterized as sensitive to various agents, including, but not limited to, IP 3 and compounds that effect the IP 3 receptor, thapsigarin, ionomycin and/or cyclic ADP-ribose (cADPR) (see, e.g. ,
  • the identities and/or cellular locations of calcium stores can be determined, for example, by isolation and characterization of organelles or imaging of cells using calcium-sensitive indicators which localize in storage organelles.
  • Mag-fura 2 an example of one such indicator, is a UV light- excitable, ratiometric, low-affinity fluorescent calcium indicator.
  • the moderate calcium affinity of mag-fura-2 and the tendency of its acetoxymethyl (AM) ester to accumulate in subcellular compartments makes this indicator particularly useful for use in monitoring of calcium stores (see, e.g., Hofer and Machen
  • Intracellular calcium stores include the endoplasmic reticulum and sarcoplasmic reticulum, which are sensitive to IP 3 or caffeine/ryanodine and thapsigarin/cyclopiazonic acid (CPA) (see, e.g., Pozzan et al. (1 994) Physiol. Rev.
  • Basal free calcium concentrations in calcium stores can be orders of magnitude, e.g., 10 3 -fold, greater than the free calcium concentration in the cytosol.
  • the basal free calcium concentration measured in the endoplasmic reticulum of HEK293 cells ranges between about 200-700 ⁇ M, with an average of about 500 ⁇ M, whereas the basal free calcium concentration in the cytosol is about 50 nM (Yu and Hinkle (2000) J. Biol. Chem. 275:23648- 23653).
  • Free calcium concentrations in the endoplasmic reticulum can be measured in a variety of ways such as, for example, using various calcium- sensitive indicators (see, e.g., Yu and Hinkle (2000) J. Biol. Chem. 275:23648- 23653) including mag-fura 2 (see, e.g., Hofer and Schulz (1996) Cell Calcium 20:235-242), endoplasmic reticulum-targeted aequorin (see, e.g., Montero et al. (1995) EMBO J.
  • various calcium- sensitive indicators see, e.g., Yu and Hinkle (2000) J. Biol. Chem. 275:23648- 23653
  • mag-fura 2 see, e.g., Hofer and Schulz (1996) Cell Calcium 20:235-242
  • endoplasmic reticulum-targeted aequorin see, e.g., Montero et al. (1995) EMBO J.
  • endoplasmic reticulum-targeted "cameleons” i.e., fluorescent calcium indicators based on fluorescence resonance energy transfer between two modified green fluorescent proteins (GFPs) contained in a protein with calmodulin and a calmodulin-binding peptide; see, e.g. , Miyawaki et al. (1 997) Nature 333:882-887 and Yu and Hinkle (2000) J. Biol. Chem. 275:23648-23653).
  • SR endoplasmic reticulum and sarcoplasmic reticulum (SR; a specialized version of the endoplasmic reticulum in striated muscle) storage organelles is achieved through sarcoplasmic-endoplasmic reticulum calcium ATPases (SERCAs), commonly referred to as calcium pumps.
  • SERCAs sarcoplasmic-endoplasmic reticulum calcium ATPases
  • endoplasmic reticulum calcium is replenished by the SERCA pump with cytoplasmic calcium that has entered the cell from the extracellular medium (Yu and Hinkle (2000) J. Biol. Chem. 275:23648-23653; Hofer et al. (1 998) EMBO J. 77:1986-1 995).
  • IP 3 receptor-mediated calcium release is triggered by IP 3 formed in the break down of plasma membrane phosphoinositides through the action of phospholipase C activated by binding of an agonist to a plasma membrane G protein-coupled receptor.
  • Ryanodine receptor-mediated calcium release is triggered by an increase in cytoplasmic calcium and is referred to as calcium-induced calcium release (CICR).
  • CICR calcium-induced calcium release
  • the activity of ryanodine receptors (which have affinity for ryanodine and caffeine) may also be regulated by cyclic ADP-ribose.
  • the calcium levels in the stores, and in the cytoplasm fluctuate.
  • ER free calcium can decrease from a range of about 60-400 ⁇ M to about 1 -50 ⁇ M when HeLa cells are treated with histamine, an agonist of PLC- linked histamine receptors (Miyawaki et al. (1 997) Nature 333:882-887) .
  • Store- operated calcium entry is activated as the free calcium concentration of the intracellular stores is reduced. Depletion of store calcium, as well as a concomitant increase in cytosolic calcium concentration, can thus regulate store- operated calcium entry into cells.
  • Store-operated ionic currents Store-operated ionic currents
  • Electrophysiological analysis of store-operated or calcium release- activated currents reveals distinct biophysical properties (see, e.g., Parekh and Penner (1997) Physiol. Rev. 77:901 -930) of these currents.
  • the current can be activated by depletion of intracellular calcium stores, and can be selective for divalent cations, such as calcium, over monovalent ions, can be influenced by changes in cytosolic calcium levels, and can show altered selectivity and conductivity in the presence of low extracellular concentrations of divalent cations.
  • proteins include, but are not limited to, calcium-binding proteins, ion transport proteins that are involved in providing for movement of cations, such as calcium, into, out of or within cells, and proteins that regulate ion transport proteins. Identification of such proteins provides specific targets that have multiple uses. For example, identification of proteins involved in modulating intracellular calcium makes possible the elucidation of the molecular and cellular mechanisms underlying such modulatory processes. Furthermore, identification of such proteins assists in the dissection of complex signalling processes and facilitates the elucidation of the element(s) involved in regulation of these processes.
  • the processes contemplated herein include, but are not limited to, receptor-mediated, store-operated, and second messenger-operated cation entry into the cytoplasm or intracellular organelles.
  • Knowledge of the number and structure of such proteins, as well as a comparison of their properties, permits the identification and design of agents that specifically interact with and/or effect or regulate proteins that modulate intracellular calcium.
  • agents have many uses. For example, they can be used to assess function and distribution of proteins that modulate intracellular calcium.
  • the specific identified proteins may also be used as targets in methods for identifying agents that modulate intracellular, such as cytosolic, calcium levels and candidate therapeutic agents.
  • the identified proteins, as well as nucleic acids encoding the proteins may be used as to modulate intracellular calcium, for example, by recombinant expression of nucleic acid encoding such a protein in a cell.
  • a protein involved in modulating intracellular calcium can do so at any level and in connection with any of a number of processes within a cell.
  • a protein involved in modulating intracellular calcium may provide for maintenance of resting cytosolic calcium levels, store-operated calcium entry into cells, receptor-mediated calcium movement, second messenger-operated calcium movement, calcium influx into or efflux from a cell, and/or calcium uptake into or release from intracellular compartments, including, for example, endosomes and lysosomes.
  • a protein involved in modulating intracellular calcium may function alone (e.g., as a single unit or as a homo-multimer of two or more of the proteins) or in combination with other proteins (e.g., in a heteromeric configuration).
  • Intracellular calcium-modulating protein identification methods involve reduction, alteration or elimination of the expression of one or more genes in a cell and assessing intracellular and/or cytosolic calcium levels and/or calcium movement into or out of the cell following reduction, alteration or elimination of the expression of one or more genes.
  • the methods are useful in identifying new proteins and/or nucleic acids encoding new proteins that were previously unknown and for identifying known proteins as intracellular calcium- modulating proteins that are involved in regulating calcium in cells.
  • Ion transport proteins are proteins involved in providing for the transport of ions into, within, or out of cells. Ion transport proteins involved in modulating intracellular calcium are involved in providing for the transport of calcium. Such ion transport proteins may be relatively specific for calcium ion transport. a. Structural features of ion transport proteins
  • An ion transport protein may function to transport cations in a number of ways.
  • the proteins may form a pore or channel for the transport of cations through a membrane.
  • the proteins may instead provide for a translocation of cations through ion binding and release processes as is characteristic of a transporter.
  • Ion transport proteins may function to transport ions as a single unit or may be one unit of a multi-component structure that transports ions.
  • a multi-component structure may be a homo-multimer of two or more of the same proteins or a hetero-multimer of two or more different proteins.
  • Transmembrane domains tend to include a sequence of amino acids, typically of about 10 to about 30 or more amino acids, about 15 to about 30 or more amino acids, about 20 to about 30 or more amino acids, or about 20 to about 25 amino acids, of high hydrophobicity.
  • the hydrophobicity scale is defined from the transfer free energy of amino acids between organic solvents and water, and statistics on the distribution of residues in proteins.
  • Pore-lining segments of ion transport protein channel transmembrane domains may have a partly hydrophilic face and appear as amphipathic segments.
  • Amphipathic segments may be identified based on the hydrophobic moment (see, e.g., Eisenberg et al. (1984) J. Mol. Biol. 773:125-142; and Finer-Moore and Stroud (1984) Proc. Natl. Acad. U.S.A. S/:155-1 59).
  • the number of transmembrane domains contained in an ion transport protein that includes a channel-like structure can vary. Typically, there are at least about 1 to about 25 or more transmembrane domains, about 2 to about 25 or more transmembrane domains, about 4 to about 25 or more transmembrane domains, about 6 to about 25 or more transmembrane domains, about 8 to about 25 or more transmembrane domains, or about 2 to about 8 transmembrane domains or about 6 or more transmembrane domains.
  • an ion transport protein that includes a channel-like structure and is capable of providing for the movement of cations through a membrane may contain at least one, or at least two, or at least three or at least 4 or more groups of six transmembrane helices.
  • Calcium transport Ion transport proteins that modulate intracellular calcium are involved in the transport of calcium. Calcium transport may be assessed in a variety ways. For example, cells expressing an ion transport protein may be evaluated for uptake of labeled calcium, such as 4S Ca 2+ , into the cells. RNA coding for an ion transport protein may also be introduced into a cell, e.g., Xenopus laevis oocytes, which may be evaluated for 45 Ca 2+ uptake.
  • Calcium transport properties of an ion transport protein may also be assessed using calcium indicator-based assays of the intracellular calcium levels of cells expressing the protein.
  • Such assays utilize calcium-sensitive indicators which facilitate detection of transient alterations in intracellular calcium levels. These indicators provide a detectable signal, e.g., fluorescence or bioluminescence, upon binding of calcium and therefore can be correlated to calcium levels in cells.
  • Methods of measuring intracellular calcium using calcium indicators are well known in the art (see, e.g., Takahashi et al. (1 999) Physiol. Rev. 73:1089-1 125).
  • Electrophysiological analysis of cells expressing an ion transport protein also can be used to assess calcium transport by a transport protein.
  • whole-cell, patch-clamp, voltage clamp and single-channel recording methods may be used to detect and measure calcium or other cation currents across membranes of cells to which calcium or another cation has been applied.
  • a variety of cells may be used for such electrophysiological analysis, including, but not limited to, X. laevis oocytes into which RNA encoding an ion transport protein has been injected.
  • Ion transport protein(s) involved in calcium regulation in cells may be more permeable to calcium than to monovalent ions under physiological conditions.
  • such types of proteins can be able to flux calcium through a membrane more readily than monovalent ions.
  • the permeability of an ion transport protein to calcium may be more than about 1 .5-fold, more than about 2-fold, more than about 3-fold, more than about 4- fold, more than about 5-fold, more than about 6-fold, more than about 7-fold, more than about 8-fold, more than about 9-fold or more than about 10-fold greater than the permeability to monovalent ions.
  • Ion transport proteins may be selective for calcium under physiological conditions.
  • the methods for identifying a protein (and/or nucleic acid encoding a protein) involved in modulating intracellular calcium involve reduction, alteration or elimination of the expression of one or more genes in a cell and the assessment of intracellular/cytosolic calcium levels and/or calcium movement into, within or out of the cells.
  • cells that are particularly suitable are those that exhibit a number of calcium transport processes and/or those in which calcium levels and/or movement may readily be assessed.
  • Another feature of cells that are particularly suitable for use in the screening and identification methods is amenability to gene expression alteration.
  • a number of techniques for altering gene expression in cells are known in the art and described herein. The relative ease with which these techniques may be applied to a cell to effect reduction, alteration or elimination of one or more genes in the cell is a consideration in selection of cells for use in the methods provided herein.
  • Amenability to gene expression alteration and analysis of ion flux particularly may be considerations, for example, when a number of genes will be screened.
  • the cells used in the method are from an organism whose genome has been extensively sequenced. Included among such organisms are Drosophlia (in particular D. me/anogaster) , Caenorhabditis (in particular, C. elegans), rodents and humans.
  • Drosophlia in particular D. me/anogaster
  • Caenorhabditis in particular, C. elegans
  • rodents rodents
  • the design of elements such as nucleic acids used in altering gene expression e.g., in antisense RNA, RNA interference and gene knock-out or knock-in methods
  • Cell type may also be a consideration when it is desired to identify an intracellular calcium-modulating protein of a particular cell type. For example, for identification of a protein involved in store-operated calcium influx in a particular cell of the central nervous system (CNS), it may be desirable to utilize a CNS cell that exhibits store-operated calcium influx in the methods.
  • CNS central nervous system
  • the cell used is one that exhibits store-operated calcium entry.
  • Such cells can be readily identified. Any method of determining the occurrence of calcium entry into a cell that distinguishes store-operated calcium entry from other types of calcium influx (e.g., entry through voltage-gated calcium channels) can be used to determine if a cell exhibits store-operated calcium entry.
  • cells may be treated to reduce the calcium levels of intracellular calcium stores and then analyzed for evidence of calcium influx in response thereto. Techniques for reducing calcium levels of intracellular stores and for analyzing cells for evidence of calcium influx are known in the art and described herein.
  • diffusible signals may be used to activate store- operated transport of calcium across the plasma membrane in methods of detecting the same.
  • One such signal is referred to as calcium influx factor (CIF) (see, e.g., Randriamampita and Tsien (1993) Nature 354:809-814; Parekh et al. (1993) Nature 364:814-818; Csutora et al. (1999) Proc. Natl. Acad. Sci. U.S.A. 36:1 21 -1 26), which may be a small ( ⁇ ⁇ 500 D) phosphate-containing anion.
  • CIF calcium influx factor
  • a CIF activity from thapsigargin-treated Jurkat cells can activate calcium influx in Xenopus oocytes and in Jurkat cells.
  • the extracts When included in the patch pipette during whole- cell patch clamp of Jurkat cells, the extracts activate an inward current resembling l CRAC .
  • electrophysiological analysis of currents across a cell- detached plasma membrane patch of a cell may be used to detect store-operated transport of calcium.
  • a variety of cell treatments may be used to reduce calcium levels in intracellular calcium stores.
  • the treatments can be viewed as either an active, direct reduction in calcium levels, such as by removal of free calcium from the stores (i.e., "active" depletion), or a passive reduction in calcium levels, such as by leak of calcium from the stores either by a reduction in the availability of free calcium for filling or replenishing the stores or by preventing filling or replenishing of stores (i.e., "passive" depletion).
  • cells may be equilibrated in 10 mM external calcium with strong buffering of cytosolic calcium, for example, through dialysis with 10 mM EGTA.
  • reduction of external calcium may also deplete intracellular calcium stores in many types of cells.
  • cells may be incubated in nominally free calcium solution, e.g., 10 ⁇ M external calcium, or essentially calcium-free solution, e.g., ⁇ 1 nM external calcium with strongly (e.g., 10 mM EGTA) buffered cytosolic calcium.
  • nominally free calcium solution e.g. 10 ⁇ M external calcium
  • essentially calcium-free solution e.g., ⁇ 1 nM external calcium with strongly (e.g., 10 mM EGTA) buffered cytosolic calcium.
  • Reduction of calcium in intracellular calcium stores can also be accomplished by application of an agent that blocks endoplasmic reticulum calcium ATPase pumps (SERCAs), thereby reducing or preventing refilling of the endoplasmic reticulum with calcium and providing for leak of calcium from the ER into the cytoplasm resulting in a reduction of ER free calcium concentration.
  • SERCAs endoplasmic reticulum calcium ATPase pumps
  • ER free calcium concentration may decrease from about 500 ⁇ M to about 50-100 ⁇ M as has been observed in HEK293 cells (Yu and Hinkle (2000) J. Biol. Chem. 275:23648-23653.
  • agents include, but are not limited to, thapsigargin, cyclopiazonic acid (CPA) and di-fert-butyl-hydroquinone (tBHQ).
  • IP 3 endoplasmic reticulum inositol-1 ,4,5-triphosphate
  • IP 3 or derivatives or analogs thereof can be provided to the endoplasmic reticulum through direct intracellular application, through application to the plasma membrane (using membrane- permeable derivatives of IP 3 ) or by contacting cells with an agent that activates the phosphoinositide cascade to generate IP 3 .
  • Such activating agents include agonists of plasma membrane receptors linked to activation of PLC and agents that activate PLC downstream of the plasma membrane in the signalling cascade.
  • G protein-coupled receptor agonists include histamine, muscarine, carbachol, substance P and glutamate. Additionally, inhibition of catabolic enzymes involved in degradation of IP 3 can serve to provide IP 3 for interaction with its receptor.
  • a membrane-permeant cation chelator that can chelate calcium within internal stores may also be used to reduce free calcium levels of the stores.
  • One such chelator is N,N,N',N'-tetrakis (2-pyridylmethyl)ethylene diamine (TPEN), which, in its uncomplexed form, diffuses across cell membranes (see, e.g., Hofer et al. (1998) J. Cell Biol. /40:325-334). Because this multivalent cation chelator has a low affinity for calcium, it should not significantly influence calcium levels in the cytoplasm or other cell compartments where the steady- state calcium concentration is in the nanomolar or low micromolar range.
  • TPEN In cell compartments where the calcium concentration is comparable to its K d , such as, for example, the endoplasmic reticulum, TPEN should bind calcium to rapidly reduce free calcium levels. Removal of TPEN from the cell medium should provide for increases in free calcium levels in such cell compartments due to rapid unbinding of the chelator from calcium ions and diffusion of the free form of TPEN from the compartment. Thus, TPEN may be used to reversibly manipulate store calcium levels without interfering with other aspects of calcium homeostasis.
  • lonophores e.g., ionomycin (a Ca 2+ /proton ionophore)
  • ionomycin a Ca 2+ /proton ionophore
  • ion flux such as occurs in store-operated calcium entry
  • patch clamp methods see, e.g., Hamil et al. (1981 ) Pflugers Arch. 337:85-100); Hoffman etal. (1 999) Nature
  • the current may be highly selective for Ca 2+ divalent ions, may display negative feedback regulation by Ca 2+ (see, e.g., Zweifach and Lewis (1995) J. Gen. Physiol. /05:209-226), and may be inhibited by divalent and trivalent metal ions such as Zn 2+ , Ni 2+ , Gd 3+ and La 3+ (Parekh and Penner (1997) Physiol. Rev. 77: 901 -930).
  • Negative feedback by Ca 2+ can be eliminated by the inclusion of very high concentrations of Ca 2+ buffers in the patch pipet. In the absence of these buffers or with buffers of lower capacity, the current may be too small to be detected, although Ca 2+ entry clearly occurs. The current may be strongly inwardly rectifying and may lose its property of inward rectification in the complete absence of divalent cations. It may be possible to determine the time course of activation of the current in single cells followed by passive store depletion via patch pipettes containing Ca 2+ chelating reagent BAPTA in the whole cell configuration using Na + used as the charge carrier (Kerschbaum and Cahalan (1999) Science 233:836).
  • the electrophysiological measurement/recording of store-operated ion flux through a membrane can provide for the assessment of such biophysiological properties as kinetics, voltage dependence, ionic selectivity.
  • the electrophysiological measurement can be performed by using whole cell patch clamp methods which can allow for the reliable and precise determination of the conditions under which Ca 2+ influx occurs. As described by Hofman et al. ((1999) Nature 337:259-263), the patch clamp technique can be used in whole- cell, cell-attached and inside-out mode.
  • patch clamp methods can be performed in a tight seal whole cell, configuration. Variations of the patch-clamp technique or other methods for detecting and analyzing ionic activity of cells, which are routine in the art, can also be used.
  • Measurement of changes of intracellular ions, such as cations including Ca 2+ can also can also be performed using fluorescence imaging, such as fluorescence videomicroscopy, digital imaging or ratioimaging techniques.
  • Measurement of changes in intracellular Ca 2+ ((Ca 2+ ),) in individual cells by fluorescence videomicroscopy can be performed using a digital imaging system, such as, for example, that produced by T.I.L.L. Photonics, or the Attofluor Digital Imaging and Photometry attachment of a Carl Zeis axiovert inverted microscope.
  • a digital imaging system such as, for example, that produced by T.I.L.L. Photonics, or the Attofluor Digital Imaging and Photometry attachment of a Carl Zeis axiovert inverted microscope.
  • cells can be grown on coverslips, rinsed and incubated with 5 ⁇ M fura2/AM (Molecular Probes) at 37 °C for 30 minutes and then washed with HPSS.
  • the coverslips with the cells are then typically clamped into a circular open-bottom chamber and mounted onto the stage of a microscope.
  • can be calculated from fluorescence ratios obtained at 340 nm and 380 nm excitation wavelengths (Garcia et. al (1994) J. Neurosci. 14:1233-1 246). Modified protocols such as those described by Zitt et. al. ((1997) J. Cell Biol. 733:1333-1341 ) and alternate forms of fluorescence ratio imaging may also be used.
  • acetoxymethyl esters which are membrane permeable.
  • the ester group is removed by cytosolic esterases, thereby trapping the free indicator in the cytosol. Interaction of the free indicator with calcium results in increased fluorescence of the indicator; therefore, an increase in the intracellular Ca 2+ concentration of cells containing the indicator can be expressed directly as an increase in fluorescence.
  • calcium flux in a cell may be monitored using a reporter gene expression system.
  • the cell in which calcium levels and fluctuations therein are monitored may contain a reporter gene encoding a detectable signal, such as luciferase, which is linked to a transcription regulatory element, e.g., promoter, that is induced by calcium-dependent factors.
  • a cell is subjected to conditions whereby the expression of one or more genes in the cell is reduced, altered or eliminated.
  • the resulting cell is analyzed to determine the effects of reduction, alteration or elimination of gene expression on intracellular calcium.
  • a protein that can modulate intracellular calcium (and nucleic acid encoding such a protein) is identified by a cell in which reduction, alteration or elimination of the expression of one or more genes is accompanied by an alteration in intracellular calcium and/or calcium ion movement into, out of or within a cell.
  • an ion transport protein can be identified by an alteration in cytosolic calcium levels and/or a reduction, alteration or elimination of store-operated ion flux into a cell upon reduction, alteration or elimination of the expression of a gene encoding the protein in the cell.
  • the methods provided herein for identifying intracellular calcium- modulating proteins (and/or nucleic acid encoding such a protein) are function- based and provide a direct correlation between a protein (and nucleic acid encoding the protein) and cell calcium, such as, for example, resting cytosolic calcium levels and/or store-operated cation flux.
  • the correlation is determined by the association of altered expression of a gene with alterations in cell calcium and/or movement of calcium into, out of or within cells.
  • particular embodiments of the methods provided herein involve identification of a gene that, when expressed in an altered fashion (including a reduction or elimination of expression), results in altered resting cytosolic calcium levels and/or altered, reduced or eliminated store-operated ion flux in a cell that contains the gene.
  • the protein encoded by the identified gene is thereby identified as an intracellular calcium-modulating protein, such as, for example, an ion transport protein, involved in maintenance of resting cytosolic calcium levels and/or store-operated calcium entry.
  • the alteration may be one that completely or nearly completely eliminates expression of a gene in the cell. If the gene is one that encodes a protein involved in maintenance of resting cytosolic calcium levels in the cell, then it may be identified, for example, by an alteration of resting cytosolic calcium levels in a cell in which expression of the gene has been eliminated. An alteration of resting cytosolic calcium levels includes increases and decreases in resting cytosolic calcium levels. If the gene is one that encodes a protein that is principally involved in providing for store-operated calcium influx in the cell, then it may be identified by a complete or nearly complete elimination of store- operated ion flux into a cell in which expression of the gene has been eliminated.
  • the gene is one that encodes a protein that substantially participates in but is not principally involved in providing for store-operated calcium influx into the cell, then complete elimination of the expression of the gene may result in an alteration and/or reduction, but perhaps not complete elimination, of store-operated ion flux into the cell.
  • a gene encodes an ion transport protein that is involved in store-operated calcium influx by being a component, such as a subunit, of a multi-subunit complex (e.g., heteromeric complex) providing for store-operated calcium influx
  • a component such as a subunit
  • a multi-subunit complex e.g., heteromeric complex
  • the alteration in gene expression may be one that only partially eliminates expression of a gene in the cell.
  • the gene may be identified by a partial reduction of store-operated ion flux into the cell.
  • the gene is one that encodes a protein that substantially participates in but is not principally involved in providing for store-operated calcium influx into the cell, then partial elimination of expression of the gene may identify the gene by an alteration and/or reduction of store- operated ion flux into the cell.
  • the methods include embodiments in which the expression of two or more genes, or a "pool" of genes, are altered in a cell.
  • a combination of the genes encodes a combination of components, each of which participates in an ion transport process that provides for store-operated calcium influx into a cell
  • complete or nearly complete elimination of expression of the combination of genes may identify the combination of genes by complete or nearly complete elimination of store-operated ion flux into the cell.
  • an "alteration" of cell calcium or calcium movement into, out of or within a cell can be a complete or nearly complete elimination of an associated activity resulting in the alteration, a reduction of the activity, an alteration in properties or characteristics of the activity or an increase in the activity.
  • an "alteration" in gene expression may be complete or nearly complete elimination of the expression of a gene, a reduction in the expression of a gene, an increase in the expression of a gene, or an alteration in the protein encoded by the gene (such as truncation or other alteration that effectively renders the protein nonfunctional or provides for aberrant functioning of the protein).
  • an alteration in gene expression may be complete or nearly complete elimination of the expression of a gene, a reduction in the expression of a gene, an increase in the expression of a gene, or an alteration in the protein encoded by the gene (such as truncation or other alteration that effectively renders the protein nonfunctional or provides for aberrant functioning of the protein).
  • RNA complementary to an RNA transcript encoded by a gene within the cell can be used to alter expression of the gene.
  • Such an approach may be referred to as an "antisense" RNA method when a single-stranded RNA molecule is introduced into a cell (see, e.g., Izant and Weintraub (1984) Cell 36:1007-1015).
  • RNA can be synthesized from, for example, phagemid clones containing DNA corresponding to a gene to be targeted for alteration of expression, using T3 and T7 polymerase. DNA templates may be removed by DNase treatments.
  • Antisense RNA is then introduced into a cell and after an appropriate period, intracellular calcium and/or calcium movement into, out of or within the cell is evaluated and compared to intracellular calcium and/or calcium movement prior to introduction of antisense RNA or to intracellular calcium and/or calcium movement in a substantially similar cell that has not received antisense RNA.
  • Antisense RNA may also be expressed in a cell by transfecting the cell with a plasmid containing nucleic acid coding for antisense RNA.
  • RNA interference is a method of gene silencing which involves the introduction of double-stranded RNA (dsRNA) into cells.
  • dsRNA double-stranded RNA
  • the basic premise of RNAi is the ability of double-stranded RNA (dsRNA) to specifically block expression of its homologous gene when present in cells.
  • dsRNA double-stranded RNA
  • a dsRNA construct containing a nucleotide sequence with homology to or identical to a portion of the target gene to be silenced is introduced into or generated within a cell containing the target gene.
  • both strands (sense and antisense) of the dsRNA are processed to small RNA fragments or segments of from about 21 -23 nucleotides (nt) in length.
  • RNAi phenomenon is mediated by a set of enzyme activities that are evolutionarily conserved in eukaryotes ranging from plants to mammals.
  • RISC nuclease multi- component nuclease
  • dsRNA fragments which may confer specificity to the nuclease through homology to the substrate mRNAs. It is believed that the dsRNA fragments instruct the RISC nuclease to destroy specific mRNAs corresponding to the dsRNA sequences.
  • An additional enzyme, Dicer has been identified that can produce the guide RNAs. Dicer is a member of the RNAse III family of nucleases that specifically cleave dsRNA and is evolutionarily conserved in worms, flies, plants, fungi and mammals.
  • the enzyme has a distinctive structure which includes a helicase domain and dual RNAse III motifs.
  • Dicer also contains a region of homology to the RDE1 /QDE2/ARGONAUTE family, which have been genetically linked to RNAi in lower eukaryotes.
  • Activation, or overexpression, of Dicer and/or Argonaute is, thus, useful for facilitating RNAi in cells, such as cultured eukaryotic cells, or mammalian cells in culture or in whole organisms.
  • cells can be treated with an agent(s) that inhibits the double- stranded RNA-dependent protein known as PKR (protein kinase RNA-activated).
  • PKR protein kinase RNA-activated
  • Part of the interferon response is the activation of the PKR response.
  • PKR phosphorylates and inactivates elF2 . Inactivation of elF2 ⁇ results in inhibition of protein synthesis and ultimately apoptosis.
  • sequence-independent PKR response can be overcome in favor of the sequence-specific RNAi response without altering the activity of PKR; however, in certain instances, it may be desirable to treat the cells with agents which inhibit expression of PKR, cause its destruction, and/or inhibit the kinase activity of PKR. Likewise, overexpression of an agent which ectopic activate elF2 ⁇ can be used.
  • the double-stranded structure may be formed by a single self- complementary RNA strand or two complementary RNA strands.
  • the dsRNA construct may include modifications to either the phosphate-sugar backbone or the nucleoside.
  • the backbone may be modified for stability or for other reasons.
  • the phosphodiester linkages may be modified to include at least one of a nitrogen or sulfur heteroatom.
  • RNA duplex formation may be initiated either inside or outside the cell.
  • the RNA may be introduced in an amount which allows delivery of at least one copy per cell. Higher doses (e.g., at least 5, 10, 100, 500, or 1000 copies per cell) of double-stranded material may yield more effective inhibition; lower doses may be useful for specific applications.
  • Inhibition is sequence-specific in that nucleotide sequences corresponding to the duplex region of the RNA are targeted for genetic inhibition. Double-stranded RNA constructs containing a nucleotide sequence identical to a portion of the target gene are generally most effective in the inhibition of target gene expression. RNA sequences with insertions, deletions, and single point mutations relative to the target sequence have also been found to be effective for inhibition.
  • Sequence identity may be optimized by sequence comparison and alignment algorithms known in the art (see e.g., Gribskov and Devereux, Sequence Analysis Primer, Stickton Press, 1991 ) and calculating the percent difference between the nucleotide sequences by, for example, the Smith-Waterman algorithm as inplemented in the BESTFIT software program using default parameters (e.g., University of Wisconsin Genetic Computing Group). Greater than 90% sequence identity, or even 100% sequence identity, generally provides for the greatest inhibition; however, it is not required for inhibition. The RNAi method is able to tolerate sequence variations that might be expected due to genetic mutation, strain polymorphism, or evolutionary divergence.
  • the duplex region of the RNA may be defined functionally as a nucleotide sequence that is capable of hybridizing with a portion of the target gene transcript.
  • the length of the identical nucleotide sequences may be, for example, at least 25, 50, 100, 200, 300, or 400 bases.
  • the dsRNA construct may be synthesized either in vivo or in vitro. Endogenous RNA polymerase of the cell may mediate transcription in vivo, or cloned RNA polymerase can be used for transcription in vivo or in vitro.
  • a regulatory region e.g., promoter, enhancer, silencer, splice donor and acceptor, polyadenylation
  • Inhibition may be targeted by specific transcription in an organ, tissue, or cell type; stimulation of an environmental condition (e.g., infection, stress, temperature, chemical inducers); and/or engineering transcription at a developmental stage or age.
  • the RNA strands may or may not be polyadenylated; the RNA strands may or may not be capable of being translated into a polypeptide by a cell's translational apparatus.
  • the dsRNA construct may be chemically or enzymatically synthesized by manual or automated reactions.
  • the dsRNA construct may be synthesized by a cellular RNA polymerase or a bacteriophage RNA polymerase (e.g., T3, T7, SP6).
  • a cellular RNA polymerase or a bacteriophage RNA polymerase e.g., T3, T7, SP6.
  • the use and production of an expression construct are known in the art (see, e.g., WO97/3201 6; U.S. Patent Nos. 5,593,874, 5,698,425, 5,71 2,135, 5,789,214, and 5,804,693).
  • the RNA may be purified prior to introduction into the cell.
  • RNA can be purified from a mixture by extraction with a solvent or resin, precipitation, electrophoresis, chromatography or a combination thereof.
  • the dsRNA construct may be used with no or a minimum of purification to avoid losses due to sample processing.
  • the dsRNA construct may be dried for storage or dissolved in an aqueous solution.
  • the solution may contain buffers or salts to promote annealing, and/or stabilization of the duplex strands.
  • RNAi can be used to alter gene expression in a cell derived from or contained in any organism.
  • the organism may be a plant, animal, protozoan, bacterium, virus, or fungus.
  • organisms include, but are not limited to Drosophila, trypanasomes, lanaria, hydra, zebrafish, Caenorhabditis elegans, mice and other mammals.
  • the cell may be from, for example, the germ line or somatic, totipotent or pluripotent, dividing or non-dividing, parenchyma or epithelium, immortalized or transformed, a stem cell or a differentiated cell.
  • the cell may be any individual cell of the early embryo, and may be a blastocyte, or, alternatively, it may be an oocyte (see, e.g., Fire et al.
  • the dsRNA may be directly injected into the cell or may be introduced by bathing the cell in a solution containing RNA.
  • Other methods for introducing dsRNA intracellularly include bombardment by particles covered by the RNA, for example gene gun technology in which the dsRNA is immobilized on gold particles and fired directly at the site, and electroporation of cell membranes in the presence of the RNA. Precise conditions for electroporation depend on the device used to produce the electro-shock and the dimensions of the chamber used to hold the cells. This method permits RNAi on a large scale. Any known gene therapy technique can also be used to administer the RNA.
  • a viral construct packaged into a viral particle would accomplish both efficient introduction of an expression construct into the cell and transcription of RNA encoded by the expression construct.
  • RNA may be introduced along with components that perform one or more of the following activities: enhance RNA uptake by the cell, promote annealing of the duplex strands, stabilize the annealed strands, or otherwise increase inhibition of the target gene.
  • a transgenic animal that expresses RNA from a recombinant construct may be produced by introducing the construct into a zygote, an embryonic stem cell, or another multipotent cell derived from the appropriate animal.
  • RNAi may also be performed on an organismal level. Mammalian cells can respond to extracellular dsRNA, and RNAi can act systemically; therefore a specific transport mechanism for dsRNA may exist (see, e.g., Asher et al. (1969) Nature 223:715-717; WO01 /36646; WO01 /68836). Consequently, injection of dsRNA into one tissue can inhibit gene function in cells throughout the animal. Thus, dsRNA may be administered extracellularly into a cavity, interstitial space, into the circulation of a mammal, introduced orally, or may be introduced by bathing an organism in a solution containing RNA.
  • Methods for oral introduction include direct mixing of the RNA with food of the organism, as well as engineered approaches in which a species that is used as food is engineered to express the RNA, then fed to the organism to be affected.
  • food bacteria such as Lactococcus lactis
  • Methods of injection include injection into vascular or extravascular circulation, the blood or lymph systems and the cerebrospinal fluid are sites where the RNA may be injected.
  • Drosophila cells are particularly well-suited for RNAi-based alteration of gene expression.
  • Many Drosophila cell lines have been established and can be biochemically characterized for use in studying various cellular processes.
  • Drosophila cell lines that are known to respond to dsRNAs by ablating expression of the target protein include S2, KC, BG2-C6, and Shi cells.
  • Many signal transduction pathways and other cellular processes have been highly conserved from Drosophila to mammals, making it possible to study complex biochemical problems in a genetically tractable model organism.
  • results obtained from the cell culture studies can be confirmed in the whole organism, because Drosophila is very amenable to RNAi analyses at the organismal level.
  • dsRNA in Drosophila cell culture to silence expression of specific genes is technically simple, efficacious, and highly reproducible.
  • the dsRNAs are efficiently internalized by the cells, thereby circumventing the problems generated by variable transfection efficiencies.
  • the gene silencing effect can be sustained through many cell divisions.
  • RNAi has been reported to achieve greater than 95% reduction in gene product. This effect can be manifested over a period of 6-7 days, thus allowing for many data points and repetition of the assay over time (Caplen et al. Gene 252:95-105 (2000)).
  • Direct gene "knock-out" procedures may also be used to alter the expression of one or more genes in a cell.
  • homologous recombination between DNA in a cell and heterologous nucleic acid introduced into the cell results in elimination of a targeted gene from the genome or alteration of the gene such that it does not produce functional protein.
  • Methods of designing nucleic acid constructs for use in targeted gene disruption or deletion are well known in the art (see, e.g., Capecchi (1 989) Science 244:1 288; Capecchi et al. (1 990) Nature 344:105; Koller et al. (1990) Science 243:1227).
  • Transfection methods may be used to introduce one or more genes into a host cell.
  • the nucleic acid(s) transferred into the host cell may encode a wild- type or altered protein or a domain, derivative, fragment or homolog thereof.
  • Transfer of nucleic acid(s) into a host cell can be accomplished by a variety of procedures. Such procedures include, but are not limited to, direct uptake using calcium phosphate (CaP0 4 ; see, e.g., Wigler et al. (1 979) Proc. Natl. Acad. Sci. U.S.A. 76:1373- 37 Q), polyethylene glycol (PEG)-mediated DNA uptake, electroporation, lipofection (see, e.g., Strauss (1 996) Meth. Mol.
  • CaP0 4 calcium phosphate
  • PEG polyethylene glycol
  • microcell fusion see, e.g., Lambert (1 991 ) Proc. Natl. Acad. Sci. U.S.A. 33:5907-591 1 ; U.S. Patent No. 5,396,767, Sawford et al. (1987) Somatic Cell Mol. Genet. /3:279-284; Dhar et al. (1 984) Somatic Cell Mol. Genet. 10:547- 559; and McNeill-Killary et al. (1995) Meth. Enzymol. 254:133-152), lipid- mediated carrier systems (see, e.g., Teifel et al.
  • nucleic acid encoding a protein of interest can be operably linked to elements that facilitate expression of the nucleic acid in host cells. Such elements include promoters, enhancers and terminators that are functional in the recipient host cell and are known to those of skill in the art.
  • the nucleic acid may be contained within a vector. Any vector known in the art for transfer and expression of nucleic acids in cells may be used, including plasmids, cosmids and artificial chromosomes.
  • more than one protein of interest may be contained on separate vectors or on the same vector in which they can be operably linked to elements that facilitate expression of the nucleic acids in host cells.
  • Multiple sequences, such as an nucleic acids expressing multiple elements in a calcium flux pathway, contained on the same vector may be controlled either by a single promoter or by multiple promoters. In a specific embodiment, the promoter is not native to the gene(s) expressing the protein(s).
  • Any methods known to those of skill in the art for the insertion of DNA fragments into a vector may be used to construct expression vectors containing a protein coding sequence of interest and appropriate transcriptional/translational control signals and/or other protein coding sequences. These methods may include in vitro recombinant DNA and synthetic techniques and in vivo recombinants (genetic recombination).
  • the cell is analyzed to evaluate the effect, if any, on intracellular calcium.
  • Intracellular calcium may be evaluated in any of a number of ways known in the art and described herein.
  • intracellular calcium can be evaluated by assessment of cytosolic or intracellular organelle calcium levels and/or fluxes or by assessment of calcium movement into or out of the cell following reduction, alteration or elimination of the expression of one or more genes.
  • Cells can be exposed to conditions (e.g., intracellular and/or extracellular calcium buffering, including use of calcium chelators, and exposure to agents that activate, inhibit or otherwise modulate various cation entry/flux processes) that facilitate assessment of intracellular calcium.
  • the resting cytosolic calcium levels, organelle calcium levels and/or store-operated calcium entry into the cell are evaluated during and/or following alteration of gene expression in a cell.
  • Resting cytosolic calcium levels, intracellular organelle calcium levels and cation movement may be assessed using any of the methods described herein or known in the art (see, e.g., descriptions herein of calcium sensitive indicator- based measurements, such as fluo-3, mag-fura 2 and ER-targeted aequorin, labelled calcium (such as 45 CA 2+ )-based measurements, and electrophysiological measurements).
  • Particular aspects of ion flux that may be assessed include, but are not limited to, a reduction (including elimination) or increase in the amount of ion flux, altered biophysical properties of the ion current, and altered sensitivities of the flux to activators or inhibitors of calcium flux processes, such as, for example, store-operated calcium entry.
  • An ion transport protein involved in store-operated calcium entry, and/or nucleic acid encoding such a protein can be identified by a cell in which reduction, alteration or elimination of the expression of a gene is accompanied by an alteration, e.g., reduction, elimination, increase or other modification, of store-operated ion flux into the cell.
  • An ion transport protein involved in maintenance of resting cytosolic calcium levels, and/or nucleic acid encoding such a protein can be identified by a cell in which reduction, alteration or elimination of the expression of a gene is accompanied by an alteration, e.g., increase, decrease or other modification, of resting cytosolic calcium levels.
  • Methods of screening for candidate intracellular calcium-modulating proteins In conducting the methods provided herein for identifying an ion transport or other intracellular calcium-modulating protein and/or nucleic acid encoding such a protein, the gene or genes that are altered in expression may be preselected, specific genes or may be random.
  • the RNA used in effecting the alteration in gene expression could be generated from a random collection of DNA or RNA sequences obtained from a cell of the type in which the alteration is being conducted.
  • the method includes alteration of the expression of one or more genes separately and/or in combinations that have been selected as candidate ion transport protein-encoding genes, calcium-binding proteins, EF hand-containing proteins or P-loop-containing proteins that may be involved in intracellular calcium modulation.
  • the cell utilized is a Drosophila cell, such as, for example, an S2 cell.
  • An embodiment of the methods of screening for candidate intracellular calcium-modulating proteins includes steps of identifying one or more genes of a cell or organism that encode(s) one or more proteins containing six or more domains, each of which has homology to an amino acid sequence characteristic of an amino acid sequence that extends through a cellular membrane (e.g., a transmembrane domain).
  • One method for evaluating the amino acid sequence of a protein for the presence of predicted transmembrane domains is through analysis using the TMpred program (www.ch.embnet.org/software/TMpred_form.html) accessed via the ExPASy Molecular Biology Server (www.expasy.ch).
  • the proteins encoded by genes of a cell or organism are evaluated for the presence of one or more domains with homology to an amino acid sequence characteristic of a protein that transports calcium, such as a calcium channel.
  • the encoded proteins may be screened only for the presence of such domains or may be screened for the presence of such domains and for the presence of multiple, e.g. , 6 or more, transmembrane domains.
  • the amino acid sequence characteristic of a protein that transports calcium can include an amino acid sequence characteristic of any one or more one of the following proteins and/or domains: ion channels, cation channels, and calcium or sodium pores.
  • the protein(s) encoded by the one or more genes do not contain one or both of the following amino acid sequences: EWKFAR and EXD(E)CR(K)GXYXXYE (wherein "X" represents any amino acid, and an amino acid residue in parentheses is an alternative to the residue immediately preceding it).
  • the proteins encoded by genes of a cell or organism are evaluated for the presence of one or more domains with homology to an amino acid sequence characteristic of a calcium-binding motif.
  • One such calcium binding motif is referred to as an EF-hand (see, e.g. , Kretsinger (1 997) Nat. Struct. Biol.
  • an EF-hand motif contains a loop of about 1 2 amino acid residues flanked on either side by an alpha helix of about 1 2 amino acid residues. Position 12 of the loop typically contains a Glu or Asp. EF-hands may undergo a conformational change upon binding calcium ions.
  • the encoded proteins may be screened only for homology to calcium-binding domains or may be screened for the homology to such domains and for the presence of multiple, e.g., 6 or more, domains with homology to transmembrane domains and/or for one or more domains having homology to an amino acid sequence characteristic of a protein that transports calcium, such as a calcium channel.
  • the proteins encoded by genes of a cell or organism are evaluated for the presence of one or more domains having homology to an amino acid sequence characteristic of a presenilin protein.
  • Presenilins are polytopic transmembrane proteins containing about six to nine transmembrane domains and a large hydrophilic loop towards the C-terminal part of the protein.
  • the encoded proteins may be screened only for homology to presenilin domains, e.g., the loop domain, or may be screened for homology to such domains and for homology to calcium-binding domains, multiple, e.g., 6 or more, transmembrane domains and/or one or more domains containing an amino acid sequence characteristic of a protein that transports calcium, such as a calcium channel.
  • the gene(s) are Drosophila, C. elegans, yeast, mammalian, mouse, rat or human genes.
  • the gene(s) are Drosophila genes.
  • Genes encoding proteins that meet the specified criteria may be identified, for example, through computer-assisted genome database searches. Such databases and search systems are known in the art. For example, a database of the Drosophila genome and system for searching genes and encoded proteins of the genome is referred to as the "GADFLY" genome annotation database of Drosophila (see hedgehog.lbl.gov and www.fruitfly.org).
  • Amino acid sequences characteristic of the selected criteria are incorporated into the annotation and search tools. Additionally, amino acid sequence pattern information is available and searchable through other databases, including InterPro (see www.ebi.ac.uk/interpro/) as well as SWISS-PROT, TrEMBL, PROSITE (see, e.g., Hofmann et al. (1999) Nucl. Acids Res. 27:215-219; Gribskov et al. (1987) Proc. Natl. Acad. Sci. U.S.A. 34:4355-4358), PRINTS, Pfam and Prodom.
  • InterPro see www.ebi.ac.uk/interpro/
  • SWISS-PROT SWISS-PROT
  • TrEMBL TrEMBL
  • PROSITE see, e.g., Hofmann et al. (1999) Nucl. Acids Res. 27:215-219; Gribskov et al. (1987) Proc. Natl. Acad. Sci
  • intracellular calcium may be evaluated in any of a number of ways, including, but not limited to, measurement of intracellular calcium levels and/or monitoring intracellular calcium level time courses.
  • Also provided herein is a method for identifying a protein involved in modulating intracellular calcium, comprising: reducing, altering or eliminating expression of one or more genes in a cell; and evaluating store-operated calcium entry into the cell, whereby one or more proteins involved in modulating intracellular calcium and/or one or more nucleic acids encoding one or more proteins involved in modulating intracellular calcium is identified in cells in which a reduction, alteration or elimination of gene expression is associated with a reduction, alteration or elimination of store- operated calcium entry in the cell; and wherein prior to reduction, alteration or elimination of gene expression, the cell exhibits store-operated calcium entry.
  • a method for identifying a protein involved in modulating intracellular calcium comprising: reducing, altering or eliminating expression of one or more genes in a cell; and evaluating cytosolic calcium levels, whereby one or more proteins involved in modulating intracellular calcium and/or one or more nucleic acids encoding one or more proteins involved in modulating intracellular calcium is identified in cells in which a reduction, alteration or elimination of gene expression is associated with an alteration in cytosolic calcium levels.
  • Another method for identifying a protein involved in modulating intracellular calcium comprises: reducing, altering or eliminating expression of one or more genes in a cell; and determining intracellular calcium levels in the resulting cell after reduction of calcium levels in an intracellular calcium store, whereby one or more proteins involved in modulating intracellular calcium and/or one or more nucleic acids encoding one or more proteins involved in modulating intracellular calcium is identified in cells in which a reduction, alteration or elimination of gene expression is associated with a reduction, alteration or elimination of an increase in cytosolic calcium levels after reduction of calcium levels of intracellular calcium stores; wherein prior to reduction, alteration or elimination of gene expression, the cell exhibits increases in cytosolic calcium levels after reduction of calcium levels of intracellular calcium stores.
  • Also provided is a method for identifying a protein involved in modulating intracellular calcium comprising: reducing, altering or eliminating expression of one or more genes in a cell; and monitoring intracellular calcium level time courses of the resulting cell after reduction of calcium levels in an intracellular calcium store, whereby one or more proteins involved in modulating intracellular calcium and/or one or more nucleic acids encoding one or more proteins involved in modulating intracellular calcium is identified in cells in which a reduction, alteration or elimination of gene expression is associated with a reduction, alteration or elimination of the fluctuations in cytosolic calcium levels after reduction of calcium levels of intracellular calcium stores; wherein prior to reduction, alteration or elimination of gene expression, the cell exhibits fluctuations in cytosolic calcium levels after reduction of calcium levels of intracellular calcium stores.
  • the protein is an ion transport protein, calcium-binding protein, EF hand-containing protein and/or a presenilin loop-containing protein.
  • the cell is a Drosophila cell, such as, for example, an S2 cell.
  • RNA comprises a sequence of nucleotides complementary to or identical to at least a portion of a gene in the cell or an RNA transcript of a gene of the cell.
  • the RNA may be double-stranded RNA.
  • any of the above methods further comprising comparing the amino acid sequence of the identified protein or proteins to amino acid sequences of proteins of species different from the species from which the cell is obtained.
  • the amino acid sequence of the identified protein or proteins is compared to mammalian protein amino acid sequences thereby identifying a mammalian protein involved in modulating intracellular calcium.
  • the mammal may be human.
  • Provided herein are any of these methods, wherein the cell exhibits increases in intracellular calcium levels after contact with thapsigargin.
  • any of the above methods for identifying a protein involved in modulating intracellular calcium wherein the one or more genes is (are) selected as targets for reduction, alteration or elimination of expression by a process comprising any one or more of the following steps:
  • the method may be conducted such that genes encoding proteins comprising one or more domains containing and/or having homology to an amino acid sequence characteristic of a protein that transports calcium are first identified and then, within that group of genes, a subset of genes is identified by evaluating those genes for encoded proteins that also comprise six or more domains each of which contains and/or has homology to an amino acid sequence characteristic of an amino acid sequence that extends through a cellular membrane.
  • the method may be conducted such that genes encoding proteins comprising one or more domains containing and/or having homology to an amino acid sequence characteristic of a protein that transports calcium are first identified and then, within that group of genes, a subset of genes is identified by evaluating those genes for encoded proteins that also comprise one or more domains containing and/or having homology to an amino acid sequence characteristic of a calcium-binding motif, and further, within that first subset of genes, a subset of genes is identified by evaluating the first subset of genes for encoded proteins that also comprise six or more domains each of which contains and/or has homology to an amino acid sequence characteristic of an amino acid sequence that extends through a cellular membrane.
  • the method may be conducted such that genes encoding proteins comprising one or more domains containing and/or having homology to an amino acid sequence characteristic of a presenilin protein, e.g., a presenilin loop sequence, are first identified and then, within that group of genes, a subset of genes is identified by evaluating those genes for encoded proteins that also comprise six or more domains each of which contains and/or has homology to an amino acid sequence characteristic of an amino acid sequence that extends through a cellular membrane.
  • genes encoding proteins comprising one or more domains containing and/or having homology to an amino acid sequence characteristic of a presenilin protein e.g., a presenilin loop sequence
  • the method may be conducted such that genes encoding proteins comprising one or more domains containing and/or having homology to an amino acid sequence characteristic of a calcium-binding motif, e.g., an EF-hand, are first identified and then, within that group of genes, a subset of genes is identified by evaluating those genes for encoded proteins that also comprise six or more domains each of which contains and/or has homology to an amino acid sequence characteristic of an amino acid sequence that extends through a cellular membrane.
  • C. Methods of Identifying an Agent that Modulates Intracellular Calcium Methods of identifying agents that modulate intracellular calcium are provided herein. The methods are based in monitoring of the effect of test agents on intracellular calcium.
  • the methods involve monitoring of resting cytosolic calcium levels and/or store-operated ion entry into cells.
  • the effect(s) of test agents on intracellular calcium can be assessed in a variety of ways including, but not limited to, evaluation of calcium or other ion (particularly cation) levels, movement of calcium or other ions (particularly cations), fluctuations in calcium or other ion (particularly cation) levels, kinetics of calcium or other ion (particularly cation) fluxes and/or transport of calcium or other ion (particularly cation) through a cellular membrane.
  • the methods are performed using cells that contain one or more proteins involved in modulation of intracellular calcium, such as, for example, ion transport proteins or calcium binding proteins, under conditions that permit evaluation of intracellular calcium.
  • the conditions permit specific evaluation of resting cytosolic calcium levels and/or store- operated calcium entry into cells.
  • the intracellular calcium-modulating protein is an ion transport protein involved in maintenance of resting cytosolic calcium levels and/or store-operated calcium entry into the cell.
  • a cell containing one or more proteins involved in modulating intracellular calcium is contacted with an agent being tested (i.e., a test agent).
  • an agent being tested i.e., a test agent.
  • Proteins involved in modulating intracellular calcium include, but are not limited to, ion transport proteins, calcium-binding proteins and regulatory proteins that regulate ion transport proteins. Proteins involved in modulating intracellular calcium include can be identified, for example, using methods as described herein. In particular embodiments of the methods for identifying agents that modulate intracellular calcium, the cell that is contacted with a test agent contains one or more proteins that are involved in maintenance of resting cytosolic calcium levels and/or store-operated calcium entry.
  • cells used in the methods of identifying agents that modulate intracellular calcium contain a protein homologous to a protein encoded by a Drosophila gene that, when altered in its expression in a Drosophila cell, results in altered store-operated calcium entry into the cell.
  • An alteration of store-operated ion flux into a cell can be a complete or nearly complete elimination of the activity, a reduction of the activity, an alteration in properties or characteristics of the activity (e.g., current properties or sensitivities) or an increase in the activity relative to the activity in a control cell (e.g., a cell such as a Drosophila cell) that has not been altered in its store- operated ion flux activity.
  • an alteration in gene expression may be complete or nearly complete elimination of the expression of a gene, a reduction in the expression of a gene, an increase in the expression of a gene, or an alteration in the protein encoded by the gene (such as truncation or other alteration that effectively renders the protein nonfunctional or provides for aberrant functioning of the protein) relative to the expression of the gene in a cell that has not been altered in its expression of the gene.
  • cells used in the methods of identifying agents that modulate intracellular calcium contain a protein homologous to a protein encoded by a Drosophila gene that, when altered in its expression in a Drosophila cell, results in altered intracellular calcium in the cell.
  • An alteration in intracellular calcium can be any alteration in calcium level, movement, location, or other calcium alteration, in a cell.
  • an alteration in intracellular calcium can be an alteration in the calcium level within an intracellular organelle or calcium storage compartment or an alteration in basal or resting cytosolic calcium levels.
  • An alteration of intracellular calcium can be any change in intracellular calcium compared to intracellular calcium in a control cell (e.g., a Drosophila cell that does not have altered expression of the gene).
  • an alteration of intracellular calcium can be, for example, an increase or decrease in basal or resting cytosolic calcium levels compared to control levels (e.g., levels in a Drosophila cell that does not have altered expression of the gene).
  • Assessment of intracellular calcium can be conducted in a number of ways, such as by methods described herein or known in the art. For example, assessment of basal or resting calcium levels can be conducted as described in Example 3 and by using any methods known in the art.
  • An alteration in basal or resting cytosolic calcium can also be effected in a number of ways, including, but not limited to, alterations in calcium flux across the plasma membrane or membranes of intracellular organelles such as endocytic organelles.
  • cells used in the methods of identifying agents that modulate intracellular calcium contain a protein homologous to a protein encoded by a Drosophila gene that, when altered in its expression in a Drosophila cell, results in altered store-operated calcium entry into the cell and altered basal or resting cytosolic calcium levels.
  • the protein homologous to a protein encoded by a Drosophila gene that, when altered in its expression in a Drosophila cell, results in altered store-operated calcium entry into the cell and/or altered intracellular calcium is at least about 20%, or at least about 25%, or at least about 30%, or least about 35%, or at least about 40%, or at least about 45%, or at least about 50%, or at least about 52%, or at least about 55%, or at least about 60%, or at least about 65%, or at least about 70%, or at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 95% or more homologous to the protein encoded by the coding sequence of the Drosophila gene.
  • the particular homology can depend on the particular protein, e.g., species, that is homologous to the Drosophila gene, the extent of the encoded Drosophila protein to which the particular protein is homologous, and can also depend on the particular Drosophila gene that, when altered in its expression in a Drosophila cell, results in altered store-operated calcium entry into the cell and/or altered intracellular calcium.
  • the protein is at least 52% or more homologous to the protein encoded by the coding sequence of the Drosophila gene.
  • Such exemplary proteins may be homologous to the specified Drosophila protein over at least about 25%, or at least about 30%, or at least about 35%, or at least about 40%, or at least about 45%, or at least about 50%, or at least about 54%, or least about 55%, or at least about 60%, or at least about 65%, or at least about 70%, or at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 95% or more of the protein encoded by the coding sequence of the Drosophila gene.
  • the protein is homologous to the Drosophila protein over at least about 54% or more of the protein encoded by the coding sequence of the Drosophila gene.
  • the cells contacted with a test agent contain one or more proteins involved in intracellular calcium modulation that is at least about 52% homologous over at least about 54% of the protein encoded by the coding sequence of the Drosophila gene.
  • cells used in the methods of identifying agents that modulate intracellular calcium contain a protein involved in modulation of intracellular calcium that is homologous to a protein encoded by the coding sequence of Drosophila gene CG8743 (Genbank Accession No. AAF491 18; gi7293750; see also SEQ ID NO. 1 for gene coding sequence and SEQ ID NO. 2 for amino acid sequence).
  • alteration, and, in particular, reduction, of the expression of CG8743 in Drosophila S2 cells is associated with a reduction in basal or resting cytosolic calcium levels and a reduction in store-operated calcium entry into the cells.
  • the protein encoded by CG8743, and proteins homologous with CG8743 are identified herein as being involved in modulating intracellular calcium.
  • such proteins are identified herein as being involved in maintenance of resting cytosolic calcium levels and/or store-operated calcium entry.
  • the cells contacted with a test agent contain one or more proteins involved in intracellular calcium modulation that has an amino acid sequence that is at least about 20%, or at least about 25%, or at least about 30%, or least about 35%, or at least about 40%, or at least about 45%, or at least about 50%, or at least about 52%, or at least about 55%, or at least about 60%, or at least about 65%, or at least about 70%, or at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 95% or more homologous to an amino acid sequence of the protein encoded by the coding sequence of at least Drosophila gene CG8743.
  • the particular homology can depend on the particular protein, e.g., species, that is homologous to the protein encoded by Drosophila gene CG8743 and the extent of the protein encoded by Drosophila gene CG8743 to which the particular protein is homologous.
  • the protein is at least 52% or more homologous to the protein encoded by the coding sequence of at least Drosophila gene CG8743.
  • Such exemplary proteins may be homologous to the specified Drosophila protein over at least about 25%, or at least about 30%, or at least about 35%, or at least about 40%, or at least about 45%, or at least about 50%, or at least about 54%, or least about 55%, or at least about 60%, or at least about 65%, or at least about 70%, or at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 95% or more of the protein encoded by the coding sequence of Drosophila gene CG8743.
  • the protein is homologous to the
  • the cells contacted with a test agent contain one or more proteins involved in intracellular calcium modulation that have an amino acid sequence that is at least about 52% homologous over at least about 54% of the protein encoded by the coding sequence of at least Drosophila gene CG8743.
  • Proteins homologous to a protein encoded by the coding sequence of Drosophila gene CG8743 include, but are not limited to, the following: Homo sapiens mucolipin-3 protein (GenBank accession No. AAL84622 (SEQ ID NO: 4); having 41 % identity and 61 % homology over 83% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO:2)); a Mus musculus mucolipin-3 (GenBank accession No.
  • AAL84623 (SEQ ID NO: 6); having 40% identity and 60% homology over 86% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO: 2)); a Mus musculus unnamed, putative mucolipidin (Genbank accession No. BAB29372 (SEQ ID NO: 8); having 39% identity and 60% homology over 78% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO: 2)); a C. elegans hypothetical protein R1 3A5.1 a (GenBank accession No.
  • AAL38964 (SEQ ID NO: 10); having 37% identity and 59% homology over 84% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO: 2)); a C. elegans hypothetical protein R13A5.1 a.p (GenBank accession No. NP_498664 (SEQ ID NO: 12); having 37% identity and 59% homology over 84% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO: 2)); a C. elegans hypothetical protein R13A5.1 b.p (GenBank accession No.
  • NP 498665 (SEQ ID NO: 14)); having 37% identity and 59% homology over 84% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO: 2)); a Homo sapiens mucolipidin protein (GenBank accession No. CAC0821 5 (SEQ ID NO: 16)); having 38% identity and 58% homology over 85% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO:2)) ; a C. elegans CUP-5L protein (GenBank accession No.
  • AAL89754 (SEQ ID NO: 18); having 37% identity and 59% homology over 84% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO: 2)); a C. elegans CUP-5 protein (GenBank accession No. AAK1 9624 (SEQ ID NO: 20); having 37% identity and 59% homology over 84% of the protein encoded by the coding sequence of Drosophila gene CG8743 SEQ ID NO: 2)); a C. elegans hypothetical protein R13A5.1 b (GenBank accession No.
  • AAL38965 (SEQ ID NO: 22); having 36% identity and 58% homology over 85% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO: 2)); a Mus musculus unnamed protein similar to mucolipin (GenBank accession No. AAH05651 (SEQ ID NO: 24); having 38% identity and 58% homology over
  • BAA91 951 (SEQ ID NO: 28); having 38% identity and 55% homology over 83% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO: 2)); a Homo sapiens mucolipidin protein (GenBank accession No. CAC07813 (SEQ ID NO: 30); having 43% identity and 63% homology over 54% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO: 2)); a Homo sapiens hypothetical protein (GenBank accession No.
  • XP 088847 (SEQ ID NO: 32); having 35% identity and 52% homology over 77% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO: 2)); a Mus musculus mucolipin-3 protein (Genbank accession No. NP 598921 (SEQ ID NO: 34); having 38% identity and 58% homology over
  • AAM28596 (SEQ ID NO: 40); having 37% identity and 58% homology over 78% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO: 2)); a Mus musculus RIKEN cDNA 3300002C04 protein (Genbank accession No. NP_080932 (SEQ ID NO: 42); having 37% identity and 58% homology over 78% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO: 2)); a Homo sapiens mucolipin 1 protein (GenBank accession No.
  • NP 065394 (SEQ ID NO 44; having 36% identity and 56% homology over 85% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO: 2)); a Mus musculus mucolipin 1 protein (GenBank accession No. NP 444407 (SEQ ID NO: 46); having 36% identity and 56% homology over 85% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO:2)); a Homo sapiens mucolipin-3 protein (GenBank accession No.
  • NP_060768 (SEQ ID NO: 48); having 36% identity and 53% homology over 83% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO 2)); a Homo sapiens mucolipidin protein (GenBank accession No. CAC07813 (SEQ ID NO: 50); having 40% identity and 59% homology over 54% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO: 2)); a C. elegans CUP-5 protein (GenBank accession No.
  • AAM1 5596 (SEQ ID NO: 52); having 35% identity and 56% homology over 84% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO: 2)); a Mus musculus mucolipin-1 protein (GenBank accession No. AAL58667 (SEQ ID NO: 54); having 36% identity and 56% homology over 85% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO: 2)); a Homo sapiens mucolipin 1 protein (GenBank accession No.
  • AAH05149 (SEQ ID NO: 56); having 36% identity and 56% homology over 85% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO: 2)); a Homo sapiens mucolipin protein (GenBank accession No. AAG00798 (SEQ ID NO: 58); having 36% identity and 56% homology over 85% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO: 2)); a Homo sapiens mucolipin protein (GenBank accession No.
  • AAG00797 (SEQ ID NO: 60); having 36% identity and 56% homology over 85% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO: 2)); a Mus musculus putative homolog to mucolipidin (GenBank accession No. BAB31730 (SEQ ID NO: 62); having 37% identity and 58% homology over 78% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO: 2)); and a Homo sapiens mucolipidosis type IV protein (GenBank accession No. AAG10422 (SEQ ID NO: 64); having 36% identity and 56% homology over 85% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO: 2)).
  • Human mucolipin-1 (MLN1 ) has been shown to be a Ca2 + -permeable channel that is transiently modulated by changes in Ca2 + concentration.
  • Mcoln2 Two mouse mucolipidins, Mcoln2 (see, GenBank Accession No. AAM08925) and Mcoln3 (GenBank Accession No. AAL84622; see, also Di Palma et al. (2002) Proc. Natl. Acad. Sci. USA 99:14994-14999), which exhibit the requiste homology to drosophila gene CG8743, are described as ion channels.
  • proteins that may be used in the methods provided herein include, but are not limited to, proteins involved in intracellular calcium modulation that are substantially homologous to the above-listed proteins.
  • the listed proteins and their sequences exemplify proteins that are useful for methods, materials and systems provided herein.
  • Naturally occurring and synthesized alternative forms of these proteins such as allelic forms, isoforms.
  • muteins, and mutated derivatives will have sequence changes that do not abolish their operation in intracellular calcium modulation and are useful in embodiments. Mutations and allelic forms of these proteins are known and are contemplated as embodiments.
  • the cells contacted with a test agent contain one or more mucolipidin (also referred to as mucolipin) or mucolipidin-like proteins.
  • Mucolipidins are membrane proteins with a predicted six transmembrane domain architecture characteristic of a channel-forming protein. Typically, mucolipidin proteins contain about 545-580 amino acids with a predicted molecular weight of about 65 kDa.
  • the first half of the human mucolipin-1 (MCOLN1 protein) contains a consensus motif of lipases with a serine active site at amino acids 104-1 14 (see, e.g., Bargal et al.
  • Mucolipidins are suggested to participate in membrane trafficking of proteins and lipids and have been implicated in transport and/or sorting in the late endocytic pathway (see, e.g., Bassi et al. (2000) Am. J. Hum. Genet. 67: 1 1 10-1 120). Potential orthologs of the mucolipidin proteins have also been identified in Mus musculus, Caenorhabditis elegans and Drosophila me/anogaster.
  • mucolipidin or mucolipidin-like proteins include the following: Homo sapiens mucolipin-3 protein (GenBank accession No. AAL84622 (SEQ ID NO: 4); having 41 % identity and 61 % homology over 83% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO: 2)); a Mus musculus mucolipin-3 (GenBank accession No. AAL84623 (SEQ ID NO: 6); having 40% identity and 60% homology over 86% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO: 2)); a Mus musculus unnamed, putative mucolipidin (GenBank accession No.
  • BAB29372 (SEQ ID NO: 8); having 39% identity and 60% homology over 78% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO: 2)); a Homo sapiens mucolipidin protein (GenBank accession No. CAC0821 5 (SEQ ID NO: 16); having 38% identity and 58% homology over 85% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO: 2)); a Mus musculus unnamed protein similar to mucolipin 1 protein (GenBank accession No.
  • AAH05651 (SEQ ID NO: 24); having 38% identity and 58% homology over 85% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO: 2)); a Homo sapiens mucolipin 1 protein (GenBank accession No. AAG42242 (SEQ ID NO: 26); having 37% identity and 56% homology over 87% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO: 2)); a Homo sapiens mucolipidin protein (GenBank accession No.
  • CAC07813 (SEQ ID NO: 30); having 43% identity and 63% homology over 54% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO: 2)); a Mus musculus mucolipin-3 protein (GenBank accession No. NP_598921 (SEQ ID NO: 34); having 38% identity and 58% homology over 86% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO: 2)); a Mus musculus mucolipin-2 protein (GenBank accession No.
  • AAM28596 (SEQ ID NO: 40); having 37% identity and 58% homology over 78% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO: 2)); a Homo sapiens mucolipin 1 protein (GenBank accession No. NP_065394 (SEQ ID NO: 44); having 36% identity and 56% homology over 85% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO: 2)); a Mus musculus mucolipin 1 protein (GenBank accession No.
  • NP 444407 SEQ ID NO: 46; having 36% identity and 56% homology over 85% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO: 2)); a Homo sapiens mucolipin-3 protein (GenBank accession No. NP 060768 (SEQ ID NO: 48); having 36% identity and 53% homology over 83% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO: 2)); a Homo sapiens mucolipidin protein (GenBank accession No.
  • CAC07813 (SEQ ID NO: 50); having 40% identity and 59% homology over 54% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO: 2)); a Mus musculus mucolipin-1 protein (GenBank accession No. AAL58667 (SEQ ID NO: 54); having 36% identity and 56% homology over 85% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO:2)); a Homo sapiens mucolipin 1 protein (GenBank accession No.
  • AAH05149 (SEQ ID NO: 56); having 36% identity and 56% homology over 85% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO: 2)); a Homo sapiens mucolipin protein (GenBank accession No. AAG00798 (SEQ ID NO: 58); having 36% identity and 56% homology over 85% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO: 2); a Homo sapiens mucolipin protein (GenBank accession No.
  • AAG00797 (SEQ ID NO: 60); having 36% identity and 56% homology over 85% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO: 2)); a Mus musculus putative homolog to mucolipidin (GenBank accession No. BAB31730 (SEQ ID NO: 62); having 37% identity and 58% homology over 78% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO: 2)); and a Homo sapiens mucolipidosis type IV protein (GenBank accession No.
  • AAG 10422 (SEQ ID NO: 64); having 36% identity and 56% homology over 85% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO: 2); human mucolipin-1 (MLN1 ; see, LaPlante et al. (2002) FEBS Lett. 532:183-187), Mcoln2 (see, GenBank Accession No. AAM08925) and Mcoln3 (GenBank Accession No. AAL84622.
  • other proteins that may be used in the methods provided herein include, but are not limited to, proteins involved in intracellular calcium modulation that are substantially homologous to the above-listed proteins.
  • Ion transport proteins are proteins involved in providing for the transport of ions, particularly cations, into, within, or out of cells.
  • the ion transport protein is one involved in providing for the movement of calcium and may be relatively specific for calcium ion transport.
  • ion transport proteins contained in the cells used in the methods are involved in maintenance of resting cytosolic calcium levels and/or store-operated calcium entry into cells, as described herein.
  • the cell contacted with a test agent contains an ion transport protein(s) containing six or more transmembrane domains.
  • the ion transport protein(s) or other protein involved in modulating intracellular calcium used in the method does not contain the following sequence of amino acids: EWKFAR.
  • This sequence of amino acids (EWKFAR) is a motif characteristic of transient receptor potential (trp) calcium channel proteins.
  • the ion transport protein(s) or other protein involved in modulating intracellular calcium used in the method does not contain the following sequence of amino acids (wherein "X" represents any amino acid, and an amino acid residue in parentheses is an alternative to the residue immediately preceding it): EXD(E)CR(K)GXYXXYE.
  • This sequence of amino acids is a motif characteristic of a pore-forming region of calcium channels such as voltage-gated calcium channels.
  • the ion transport protein(s) or other protein involved in modulating intracellular calcium used in the method does not contain either of the above- mentioned amino acid sequences, i.e., EWKFAR or EXD(E)CR(K)GXYXXYE. d. Cells
  • Any cell that contains one or more proteins involved in modulating intracellular calcium as described above may be used in the methods of identifying agents that modulate intracellular calcium.
  • Particular cells include cells containing one or more proteins that are involved in maintenance of resting cytosolic calcium levels and/or store-operated calcium entry.
  • Particularly suitable cells include any cell which is amenable to monitoring of intracellular calcium, such as, for example, monitoring of resting cytosolic calcium levels and/or store- operated calcium entry or in which store-operated calcium entry occurs or that can be manipulated such that store-operated calcium entry occurs in the cell.
  • the cell may endogenously express the protein(s) involved in modulating intracellular calcium or recombinantly express the protein(s) through introduction of heterologous nucleic acid encoding the protein(s) into the cell using methods known in the art and described herein.
  • the cell is a recombinant cell that expresses the protein(s) as heterologous protein(s). Such cells may overexpress the heterologous protein(s).
  • a recombinant cell may be one that endogenously expresses the protein(s) and also has been transfected with additional copies of nucleic acid encoding the protein(s).
  • the host cell used in generating the recombinant cell can be one that endogenously expresses little to no store-operated calcium entry activity, e.g., CHO-K1 cells, or a host cell in which endogenous store-operated calcium entry activity has been eliminated (e.g., through gene knock-out methods or by inhibition with an agent that does not inhibit store-operated calcium entry activity of the heterologous protein(s)).
  • Cells or less differentiated precursor cells having an endogenous protein involved in modulating intracellular calcium can be used.
  • Cells or less differentiated precursor cells may be recombinant cells stably or transiently transfected with nucleic acid encoding intracellular calcium-modulating protein(s) in vitro or in an organism. In vitro transfection can be followed by cell expansion through culturing prior to use. .
  • Cells for use in the methods may be of any species.
  • the cells can be eukaryotic cells.
  • the cells can be mammalian cells. Mammalian cells include, but are not limited to, rodent (e.g., mouse, rat and hamster), primate, monkey, dog, bovine, rabbit and human cells.
  • rodent e.g., mouse, rat and hamster
  • primate monkey, dog, bovine, rabbit and human cells.
  • neuronal, nervous system e.g., CNS
  • tissue-derived or brain cells that contain (endogenously and/or recombinantly) one or more proteins involved in modulating intracellular calcium as described above may be used in the methods of identifying agents that modulate intracellular calcium.
  • Cells from a known cell line can be used, such as from neuroblastoma SH-SY5Y cells, pheochromocytoma PC 1 2 cells, neuroblastoma SK-N-BE(2)C cells, human SK-N-MC neuroblastoma cells, SMS-KCNR cells, human LAN-5 neuroblastoma cells, human GI-CA-N neuroblatoma cells, human GOTO neuroblastoma cells, mouse Neuro 2a (N2A) neuroblastoma cells and/or human IMR 32 neuroblastoma cells.
  • Cell lines include HEK 293, CHO (including CHO-K1 ), LTK " , N2A, H6, and HGB. The generation, maintenance and use of such cell lines is well known.
  • the host cell for generation of a recombinant cell may be a less differentiated precursor cell, and preferably is one that is readily stably transfected.
  • test agents on intracellular calcium
  • intracellular calcium and/or movement of ions into, out of or within the cell is evaluated.
  • the effect of a test agent on intracellular calcium is determined by comparing intracellular calcium and/or movement of ions into, out of or within the cell upon or after contacting the cell with the agent to intracellular calcium and/or the movement of ions into, out of or within a control cell.
  • One control cell is a cell substantially identical to, if not the same as, the cell that is contacted with the test agent, but is such cell in the absence of the test agent.
  • Another control cell is one that is substantially similar cell to the test cell (i.e., the cell containing one or more proteins involved in modulating intracellular calcium) but that does not contain the particular one or more proteins involved in modulating intracellular calcium.
  • the test cell containing the one or more proteins involved in modulating intracellular calcium is a recombinant cell generated by transfer of nucleic acid encoding the one or more proteins into a host cell
  • one possible control cell is a host cell that has not been transfected with nucleic acid encoding the one or more proteins.
  • Such a cell would be substantially similar to the test cell but would differ from the test cell essentially only by the absence of the introduced nucleic acid encoding the one or more proteins.
  • any aspect of intracellular calcium can be evaluated in the methods for identifying agents that modulate intracellular calcium.
  • any one or more of the following aspects may be evaluated: maintenance of resting cytosolic calcium levels, store-operated ion entry into cells, receptor-mediated ion (e.g., calcium) movement, second messenger-operated ion (e.g., calcium) movement, calcium influx into or efflux out of the cell, and/or ion (e.g., calcium) uptake into or release from intracellular compartments, including, for example, endosomes and lysosomes.
  • the evaluation can be conducted using any methods described herein or known in the art for evaluating intracellular calcium and/or movement of ions into, out of or within cells. In particular embodiments of the methods for identifying agents that modulate intracellular calcium, basal or resting calcium levels and/or store-operated ion entry are evaluated.
  • test agents In methods for identifying agents that modulate intracellular calcium that include a step of evaluating store-operated calcium entry in cells contacted with test agent, the methods are conducted under conditions that permit store- operated calcium entry to occur. Such conditions are described herein and are known in the art. Test agents are brought into contact with the cells used in the methods under these appropriate conditions.
  • test agent is identified as an agent that modulates intracellular calcium if intracellular calcium and/or movement of ions into, out of or within the cell upon or after contacting the cell with the agent differs from intracellular calcium and/or the movement of ions into, out of or within a control cell.
  • the difference in intracellular calcium and/or the movement of ions into, out of or within a test and control cell can be any detectable difference in intracellular calcium determined in a number of ways.
  • a detectable or measurable difference in any one or more of the following aspects of intracellular calcium between a test and control cell can be used to identify a test agent as an agent that modulates intracellular calcium: resting cytosolic calcium levels, store- operated ion entry into the cells, receptor-mediated ion (e.g., calcium) movement, second messenger-operated ion (e.g., calcium) movement, calcium influx into or efflux out of a cell, and/or ion (e.g., calcium) uptake into or release from intracellular compartments, including, for example, endosomes and lysosomes.
  • receptor-mediated ion e.g., calcium
  • second messenger-operated ion e.g., calcium
  • ion e.g., calcium
  • test and control cells can be conducted using any methods described herein or known in the art for evaluating intracellular calcium and/or movement of ions into, out of or within cells.
  • a test agent is identified as an agent that modulates intracellular calcium if basal or resting calcium levels and/or store-operated ion entry in the test cell differs from that in the control cell.
  • Systems Also provided herein are systems for use in identifying an agent that modulates intracellular, e.g., cytoplasmic, calcium.
  • Such systems include a cell containing one or more proteins involved in intracellular calcium modulation that has an amino acid sequence homologous to an amino acid sequence of a protein encoded by a Drosophila gene that, when altered in its expression in a Drosophila cell, results in altered store-operated calcium entry into the cell and/or altered intracellular calcium, e.g., basal or resting cytosolic calcium levels.
  • the one or more proteins has an amino acid sequence that is at least about 20%, or at least about 25%, or at least about 30%, or least about 35%, or at least about 40%, or at least about 45%, or at least about 50%, or at least about 52%, or at least about 55%, or at least about 60%, or at least about 65%, or at least about 70%, or at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 95% or more homologous to an amino acid sequence of the protein encoded by the coding sequence of the Drosophila gene.
  • the particular homology can depend on the particular protein, e.g., species, that is homologous to the Drosophila gene, the extent of the encoded Drosophila protein to which the particular protein is homologous, and can also depend on the particular Drosophila gene that, when altered in its expression in a Drosophila cell, results in altered store- operated calcium entry into the cell and/or altered intracellular calcium.
  • the protein is at least 52% or more homologous to the protein encoded by the coding sequence of the Drosophila gene.
  • Such exemplary proteins may be homologous to the specified Drosophila protein over at least about 25%, or at least about 30%, or at least about 35%, or at least about 40%, or at least about 45%, or at least about 50%, or at least about 54%, or least about 55%, or at least about 60%, or at least about 65%, or at least about 70%, or at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 95% or more of the protein encoded by the coding sequence of the Drosophila gene.
  • the protein is homologous to the Drosophila protein over at least about 54% or more of the protein encoded by the coding sequence of the Drosophila gene.
  • the cell contains one or more proteins involved in intracellular calcium modulation that is at least about 52% homologous over at least about 54% of the protein encoded by the coding sequence of the Drosophila gene.
  • the cell contains one or more proteins involved in modulation of intracellular calcium that is homologous to a protein encoded by the coding sequence of Drosophila gene CG8743 (Genbank Accession No. AAF491 18; gi7293750; see also SEQ ID NO. 1 for gene coding sequence and SEQ ID NO. 2 for amino acid sequence).
  • a system provided herein includes a cell containing one or more proteins involved in intracellular calcium modulation that has an amino acid sequence that is at least about 20%, or at least about 25%, or at least about 30%, or least about 35%, or at least about 40%, or at least about 45%, or at least about 50%, or at least about 52%, or at least about 55%, or at least about 60%, or at least about 65%, or at least about 70%, or at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 95% or more homologous to an amino acid sequence of the protein encoded by the coding sequence of at least Drosophila gene CG8743.
  • the particular homology can depend on the particular protein, e.g., species, that is homologous to the protein encoded by Drosophila gene CG8743 and the extent of the protein encoded by Drosophila gene CG8743 to which the particular protein is homologous.
  • the protein is at least 52% or more homologous to the protein encoded by the coding sequence of at least Drosophila gene CG8743.
  • Such exemplary proteins may be homologous to the specified Drosophila protein over at least about 25%, or at least about 30%, or at least about 35%, or at least about 40%, or at least about 45%, or at least about 50%, or at least about 54%, or least about 55%, or at least about 60%, or at least about 65%, or at least about 70%, or at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 95% or more of the protein encoded by the coding sequence of Drosophila gene CG8743.
  • the protein is homologous to the Drosophila protein over at least about 54% or more of the protein encoded by the coding sequence of CG8743.
  • the protein has an amino acid sequence that is at least about 52% homologous over at least about 54% of the protein encoded by the coding sequence of at least Drosophila gene CG8743.
  • Proteins homologous to a protein encoded by the coding sequence of Drosophila gene CG8743 include, but are not limited to, the following: Homo sapiens mucolipin-3 protein (GenBank accession No. AAL84622 (SEQ ID NO: 4); having 41 % identity and 61 % homology over 83% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO:2)); a Mus musculus mucolipin-3 (GenBank accession No.
  • AAL84623 (SEQ ID NO: 6); having 40% identity and 60% homology over 86% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO: 2)); a Mus musculus unnamed, putative mucolipidin (Genbank accession No. BAB29372 (SEQ ID NO: 8); having 39% identity and 60% homology over 78% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO: 2)); a C. elegans hypothetical protein R1 3A5.1 a (GenBank accession No.
  • AAL38964 (SEQ ID NO: 10); having 37% identity and 59% homology over 84% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO: 2)); a C. elegans hypothetical protein R1 3A5.1 a.p (GenBank accession No. NP 498664 (SEQ ID NO: 12); having 37% identity and 59% homology over 84% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO: 2)); a C. elegans hypothetical protein R1 3A5.1 b.p (GenBank accession No.
  • NP_498665 (SEQ ID NO: 14)); having 37% identity and 59% homology over 84% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO: 2)); a Homo sapiens mucolipidin protein (GenBank accession No. CAC0821 5 (SEQ ID NO: 16)); having 38% identity and 58% homology over 85% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO:2)) ; a C. elegans CUP-5L protein (GenBank accession No.
  • AAL89754 (SEQ ID NO: 18); having 37% identity and 59% homology over 84% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO: 2)); a C. elegans CUP-5 protein (GenBank accession No. AAK1 9624 (SEQ ID NO: 20); having 37% identity and 59% homology over 84% of the protein encoded by the coding sequence of Drosophila gene CG8743 SEQ ID NO: 2)); a C. elegans hypothetical protein R13A5.1 b (GenBank accession No.
  • AAL38965 (SEQ ID NO: 22); having 36% identity and 58% homology over 85% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO: 2)); a Mus musculus unnamed protein similar to mucolipin (GenBank accession No.
  • AAH05651 (SEQ ID NO: 24); having 38% identity and 58% homology over 85% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO: 2)); a Homo sapiens mucolipin 1 protein (GenBank accession No. AAG42242 (SEQ ID NO: 26); having 37% identity and 56% homology over 87% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO: 2)); a Homo sapiens unnamed protein product (GenBank accession No.
  • BAA91951 (SEQ ID NO: 28); having 38% identity and 55% homology over 83% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO: 2)); a Homo sapiens mucolipidin protein (GenBank accession No. CAC07813 (SEQ ID NO: 30); having 43% identity and 63% homology over 54% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO: 2)); a Homo sapiens hypothetical protein (GenBank accession No.
  • XP 088847 (SEQ ID NO: 32); having 35% identity and 52% homology over 77% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO: 2)); a Mus musculus mucolipin-3 protein (Genbank accession No. NP 598921 (SEQ ID NO: 34); having 38% identity and 58% homology over 86% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO: 2)); a C. elegans CUP-5 protein (GenBank accession No.
  • AAM1 5598 (SEQ ID NO: 36); having 35% identity and 56% homology over 84% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO: 2)); a C. elegans CUP-5 protein (Genbank accession No. AAM15597 (SEQ ID NO: 38); having 35% identity and 56% homology over
  • NP_080932 (SEQ ID NO: 42); having 37% identity and 58% homology over 78% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO: 2)); a Homo sapiens mucolipin 1 protein (GenBank accession No. NP_065394 (SEQ ID NO 44; having 36% identity and 56% homology over 85% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO: 2)); a Mus musculus mucolipin 1 protein (GenBank accession No.
  • NP_444407 SEQ ID NO: 46; having 36% identity and 56% homology over 85% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO:2)); a Homo sapiens mucolipin-3 protein (GenBank accession No. NP_060768 (SEQ ID NO: 48); having 36% identity and 53% homology over 83% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO 2)); a Homo sapiens mucolipidin protein (GenBank accession No.
  • CAC0781 3 (SEQ ID NO: 50); having 40% identity and 59% homology over 54% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO: 2)); a C. elegans CUP-5 protein (GenBank accession No. AAM15596 (SEQ ID NO: 52); having 35% identity and 56% homology over 84% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO: 2)); a Mus musculus mucolipin-1 protein (GenBank accession No.
  • AAL58667 (SEQ ID NO: 54); having 36% identity and 56% homology over 85% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO: 2)); a Homo sapiens mucolipin 1 protein (GenBank accession No. AAH05149 (SEQ ID NO: 56); having 36% identity and 56% homology over 85% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO: 2)); a Homo sapiens mucolipin protein (GenBank accession No.
  • AAG00798 (SEQ ID NO: 58); having 36% identity and 56% homology over 85% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO: 2)); a Homo sapiens mucolipin protein (GenBank accession No. AAG00797 (SEQ ID NO: 60); having 36% identity and 56% homology over 85% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO: 2)); a Mus musculus putative homolog to mucolipidin (GenBank accession No.
  • BAB31730 (SEQ ID NO: 62); having 37% identity and 58% homology over 78% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO: 2)); and a Homo sapiens mucolipidosis type IV protein (GenBank accession No. AAG10422 (SEQ ID NO: 64); having 36% identity and 56% homology over 85% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO: 2)).
  • other proteins that may be used in the methods provided herein include, but are not limited to, proteins involved in intracellular calcium modulation that are substantially homologous to the above-listed proteins.
  • the system in another embodiment, includes a cell containing one or more mucolipidin (also referred to as mucolipin) or mucolipidin-like proteins.
  • mucolipidin also referred to as mucolipin
  • mucolipidin-like proteins include the following: Homo sapiens mucolipin-3 protein (GenBank accession No. AAL84622 (SEQ ID NO: 4); having 41 % identity and 61 % homology over 83% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO: 2)); a Mus musculus mucolipin-3 (GenBank accession No.
  • AAL84623 (SEQ ID NO: 6); having 40% identity and 60% homology over 86% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO: 2)); a Mus musculus unnamed, putative mucolipidin (GenBank accession No. BAB29372 (SEQ ID NO: 8); having 39% identity and 60% homology over 78% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO: 2)); a Homo sapiens mucolipidin protein (GenBank accession No.
  • CAC08215 (SEQ ID NO: 16); having 38% identity and 58% homology over 85% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO: 2)); a Mus musculus unnamed protein similar to mucolipin 1 protein (GenBank accession No. AAH05651 (SEQ ID NO: 24); having 38% identity and 58% homology over 85% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO: 2)); a Homo sapiens mucolipin 1 protein (GenBank accession No.
  • AAG42242 (SEQ ID NO: 26); having 37% identity and 56% homology over 87% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO: 2)); a Homo sapiens mucolipidin protein (GenBank accession No. CAC07813 (SEQ ID NO: 30); having 43% identity and 63% homology over 54% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO: 2)); a Mus musculus mucolipin-3 protein (GenBank accession No.
  • NP_598921 (SEQ ID NO: 34); having 38% identity and 58% homology over 86% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO: 2)); a Mus musculus mucolipin-2 protein (GenBank accession No. AAM28596 (SEQ ID NO: 40); having 37% identity and 58% homology over 78% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO: 2)); a Homo sapiens mucolipin 1 protein (GenBank accession No.
  • NP 065394 (SEQ ID NO: 44); having 36% identity and 56% homology over 85% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO: 2)); a Mus musculus mucolipin 1 protein (GenBank accession No. NP 444407 (SEQ ID NO: 46); having 36% identity and 56% homology over 85% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO: 2)); a Homo sapiens mucolipin-3 protein (GenBank accession No.
  • NP_060768 (SEQ ID NO: 48); having 36% identity and 53% homology over 83% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO: 2)); a Homo sapiens mucolipidin protein (GenBank accession No. CAC0781 3 (SEQ ID NO: 50); having 40% identity and 59% homology over 54% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO: 2)); a Mus musculus mucolipin-1 protein (GenBank accession No.
  • AAL58667 (SEQ ID NO: 54); having 36% identity and 56% homology over 85% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO:2)); a Homo sapiens mucolipin 1 protein (GenBank accession No. AAH05149 (SEQ ID NO: 56); having 36% identity and 56% homology over 85% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO: 2)); a Homo sapiens mucolipin protein (GenBank accession No.
  • AAG00798 (SEQ ID NO: 58); having 36% identity and 56% homology over 85% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO: 2); a Homo sapiens mucolipin protein (GenBank accession No. AAG00797 (SEQ ID NO: 60); having 36% identity and 56% homology over 85% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO: 2)); a Mus musculus putative homolog to mucolipidin (GenBank accession No.
  • BAB31 730 (SEQ ID NO: 62); having 37% identity and 58% homology over 78% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO: 2)); and a Homo sapiens mucolipidosis type IV protein (GenBank accession No. AAG10422 (SEQ ID NO: 64); having 36% identity and 56% homology over 85% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO: 2).
  • other proteins that may be used in the methods provided herein include, but are not limited to, proteins involved in intracellular calcium modulation that are substantially homologous to the above-listed proteins.
  • the cells of the system can be isolated cells or cell cultures that endogenously express such protein(s) or can recombinantly express such proteins as described above with respect to the methods for identifying agents.
  • Systems in which the cells recombinantly express the proteins can be such that the cells are isolated cells or cell cultures or are contained within an animal, in particular, a non-human animal, e.g., a non-human mammal.
  • the cells of the system can be contained in a medium that contains an agent that provides for passive or active intracellular calcium store reduction or depletion and/or that contains a molecule or molecules that facilitate monitoring or measurement of intracellular calcium and/or calcium movement.
  • Such molecules include fluorescent (or otherwise labeled) calcium indicators, trivalent cations, divalent cations other than calcium and calcium-buffering agents, e.g., calcium chelators.
  • Proteins involved in modulating intracellular calcium further provide the basis for additional methods of identifying molecules involved in modulating intracellular calcium, as well as for methods of elucidating pathways, and elements thereof, of intracellular calcium modulation.
  • a protein Once a protein has been identified as one involved in modulating intracellular calcium, it can be used to identify molecules, in particular cellular components, that interact with it and function in the modulation of intracellular calcium. Additionally, the identification of molecules that interact with proteins involved in intracellular calcium modulation, facilitates the dissection and elucidation of pathways and mechanisms of cellular calcium regulation and signalling. The elucidation of such pathways provides additional targets that can be modulated in methods of modulating intracellular calcium and for use in methods of identifying agents for modulating intracellular calcium.
  • a protein involved in intracellular calcium modulation is the protein encoded by Drosophila gene CG8743.
  • alteration, and, in particular, reduction, of the expression of CG8743 in Drosophila S2 cells is associated with a reduction in basal or resting cytosolic calcium levels and a reduction in store-operated calcium entry into the cells.
  • the protein encoded by CG8743 is involved in maintenance of resting cytosolic calcium levels and/or store-operated calcium entry.
  • Proteins homologous to the protein encoded by the CG8743 gene including, for example, mucolipidin and mucolipidin-like proteins, that modulate intracellular calcium can thus be used in methods to identify molecules, in particular cellular components, that interact with the proteins and that may be involved in modulating intracellular calcium.
  • the method includes a step of assaying for a molecule, such as, for example, a cellular component, that interacts with the protein encoded by Drosophila gene CG8743.
  • a molecule such as, for example, a cellular component
  • the candidate molecule can then be further tested for intracellular calcium-modulating properties by evaluating the effect of modulating the molecule and/or its interaction with the protein encoded by Drosophila gene CG8743 on intracellular calcium.
  • the method includes a step of assaying for a molecule, such as, for example, a cellular component, that interacts with a protein that is homologous to the protein encoded by Drosophila gene CG8743.
  • a molecule such as, for example, a cellular component
  • the protein that is homologous to the protein encoded by Drosophila gene CG8743 has an amino acid sequence that is at least about 20%, or at least about 25%, or at least about 30%, or least about 35%, or at least about 40%, or at least about 45%, or at least about 50%, or at least about 52%, or at least about 55%, or at least about 60%, or at least about 65%, or at least about 70%, or at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 95% or more homologous to an amino acid sequence of the protein encoded by the coding sequence of Drosophila gene CG8743.
  • the particular homology can depend on the particular protein, e.g., species, that is homologous to the protein encoded by Drosophila gene CG8743 and the extent of the protein encoded by Drosophila gene CG8743 to which the particular protein is homologous.
  • the protein is at least 52% or more homologous to the protein encoded by the coding sequence of Drosophila gene CG8743.
  • Such exemplary proteins may be homologous to the protein encoded by the coding sequence of Drosophila gene CG8743 over at least about 25%, or at least about 30%, or at least about 35%, or at least about 40%, or at least about 45%, or at least about 50%, or at least about 54%, or least about 55%, or at least about 60%, or at least about 65%, or at least about 70%, or at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 95% or more of the protein encoded by the coding sequence of Drosophila gene CG8743.
  • the protein is homologous to the protein encoded by the coding sequence of Drosophila gene CG8743 over at least about 54% or more of the protein encoded by the coding sequence of CG8743.
  • the method includes a step of assaying for a molecule, such as, for example, a cellular component, that interacts with a protein that has an amino acid sequence that is at least about 52% homologous over at least about 54% of the protein encoded by the coding sequence of Drosophila gene CG8743.
  • a molecule such as, for example, a cellular component
  • Proteins homologous to a protein encoded by the coding sequence of Drosophila gene CG8743 include, but are not limited to, the following: Homo sapiens mucolipin-3 protein (GenBank accession No. AAL84622 (SEQ ID NO: 4); having 41 % identity and 61 % homology over 83% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO:2»; a Mus musculus mucolipin-3 (GenBank accession No.
  • AAL84623 (SEQ ID NO: 6); having 40% identity and 60% homology over 86% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO: 2)); a Mus musculus unnamed, putative mucolipidin (Genbank accession No.
  • BAB29372 (SEQ ID NO: 8); having 39% identity and 60% homology over 78% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO: 2)); a C. elegans hypothetical protein R13A5.1 a (GenBank accession No. AAL38964 (SEQ ID NO: 10); having 37% identity and 59% homology over 84% of the protein encoded by the coding sequence of
  • Drosophila gene CG8743 (SEQ ID NO: 2)); a C. elegans hypothetical protein R1 3A5.1 a.p (GenBank accession No. NP 498664 (SEQ ID NO: 1 2); having 37% identity and 59% homology over 84% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO: 2)); a C. elegans hypothetical protein R13A5.1 b.p (GenBank accession No.
  • NP_498665 (SEQ ID NO: 14)); having 37% identity and 59% homology over 84% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO: 2)); a Homo sapiens mucolipidin protein (GenBank accession No. CAC0821 5 (SEQ ID NO: 16)); having 38% identity and 58% homology over 85% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO:2)) ; a C. elegans CUP-5L protein (GenBank accession No.
  • AAL89754 (SEQ ID NO: 18); having 37% identity and 59% homology over 84% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO: 2)); a C. elegans CUP-5 protein (GenBank accession No. AAK19624 (SEQ ID NO: 20); having 37% identity and 59% homology over 84% of the protein encoded by the coding sequence of Drosophila gene CG8743 SEQ ID NO: 2)); a C. elegans hypothetical protein R13A5.1 b (GenBank accession No.
  • AAL38965 (SEQ ID NO: 22); having 36% identity and 58% homology over 85% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO: 2)); a Mus musculus unnamed protein similar to mucolipin (GenBank accession No. AAH05651 (SEQ ID NO: 24); having 38% identity and 58% homology over 85% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO: 2)); a Homo sapiens mucolipin 1 protein (GenBank accession No.
  • AAG42242 (SEQ ID NO: 26); having 37% identity and 56% homology over 87% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO: 2)); a Homo sapiens unnamed protein product (GenBank accession No. BAA91951 (SEQ ID NO: 28); having 38% identity and 55% homology over 83% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO: 2)); a Homo sapiens mucolipidin protein (GenBank accession No.
  • CAC07813 (SEQ ID NO: 30); having 43% identity and 63% homology over 54% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO: 2)); a Homo sapiens hypothetical protein (GenBank accession No. XP_088847 (SEQ ID NO: 32); having 35% identity and 52% homology over 77% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO: 2)); a Mus musculus mucolipin-3 protein (Genbank accession No.
  • NP_598921 (SEQ ID NO: 34); having 38% identity and 58% homology over 86% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO: 2)); a C. elegans CUP-5 protein (GenBank accession No. AAM1 5598 (SEQ ID NO: 36); having 35% identity and 56% homology over
  • AAM28596 (SEQ ID NO: 40); having 37% identity and 58% homology over 78% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO: 2)); a Mus musculus RIKEN cDNA 3300002C04 protein (Genbank accession No. NP 080932 (SEQ ID NO: 42); having 37% identity and 58% homology over 78% of the protein encoded by the coding sequence of
  • Drosophila gene CG8743 (SEQ ID NO: 2)); a Homo sapiens mucolipin 1 protein (GenBank accession No. NP_065394 (SEQ ID NO 44; having 36% identity and 56% homology over 85% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO: 2)); a Mus musculus mucolipin 1 protein (GenBank accession No. NP_444407 (SEQ ID NO: 46); having 36% identity and 56% homology over 85 % of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO:2)); a Homo sapiens mucolipin-3 protein (GenBank accession No.
  • NP 060768 SEQ ID NO: 48; having 36% identity and 53% homology over 83% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO 2)); a Homo sapiens mucolipidin protein (GenBank accession No. CAC0781 3 (SEQ ID NO: 50); having 40% identity and 59% homology over 54% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO: 2)); a C. elegans CUP-5 protein (GenBank accession No. AAM1 5596 (SEQ ID NO: 52); having 35% identity and 56% homology over 84% of the protein encoded by the coding sequence of
  • Drosophila gene CG8743 (SEQ ID NO: 2)); a Mus musculus mucolipin-1 protein (GenBank accession No. AAL58667 (SEQ ID NO: 54); having 36% identity and 56% homology over 85% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO: 2)); a Homo sapiens mucolipin 1 protein (GenBank accession No. AAH05149 (SEQ ID NO: 56); having 36% identity and 56% homology over 85% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO: 2)); a Homo sapiens mucolipin protein (GenBank accession No.
  • AAG00798 (SEQ ID NO: 58); having 36% identity and 56% homology over 85% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO: 2)); a Homo sapiens mucolipin protein (GenBank accession No. AAG00797 (SEQ ID NO: 60); having 36% identity and 56% homology over 85% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO: 2)); a Mus musculus putative homolog to mucolipidin (GenBank accession No.
  • BAB31730 (SEQ ID NO: 62); having 37% identity and 58% homology over 78% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO: 2)); and a Homo sapiens mucolipidosis type IV protein (GenBank accession No. AAG 10422 (SEQ ID NO: 64); having 36% identity and 56% homology over 85% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO: 2)).
  • other proteins that may be used in the methods provided herein include, but are not limited to, proteins involved in intracellular calcium modulation that are substantially homologous to the above-listed proteins.
  • the method includes a step of assaying for a molecule, such as, for example, a cellular component, that interacts with a mucolipidin (also referred to as mucolipin) or mucolipidin-like protein.
  • a mucolipidin also referred to as mucolipin
  • mucolipidin-like proteins include the following: Homo sapiens mucolipin-3 protein (GenBank accession No. AAL84622 (SEQ ID NO: 4); having 41 % identity and 61 % homology over 83% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO: 2)); a Mus musculus mucolipin-3 (GenBank accession No.
  • AAL84623 (SEQ ID NO: 6); having 40% identity and 60% homology over 86% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO: 2)); a Mus musculus unnamed, putative mucolipidin (GenBank accession No. BAB29372 (SEQ ID NO: 8); having 39% identity and 60% homology over 78% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO: 2)); a Homo sapiens mucolipidin protein (GenBank accession No.
  • CAC0821 5 (SEQ ID NO: 16); having 38% identity and 58% homology over 85% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO: 2)); a Mus musculus unnamed protein similar to mucolipin 1 protein (GenBank accession No. AAH05651 (SEQ ID NO: 24); having 38% identity and 58% homology over 85% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO: 2)); a Homo sapiens mucolipin 1 protein (GenBank accession No.
  • AAG42242 (SEQ ID NO: 26); having 37% identity and 56% homology over 87% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO: 2)); a Homo sapiens mucolipidin protein (GenBank accession No. CAC07813 (SEQ ID NO: 30); having 43% identity and 63% homology over 54% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO: 2)); a Mus musculus mucolipin-3 protein (GenBank accession No.
  • NP 598921 (SEQ ID NO: 34); having 38% identity and 58% homology over 86% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO: 2)); a Mus musculus mucolipin-2 protein (GenBank accession No. AAM28596 (SEQ ID NO: 40); having 37% identity and • 58% homology over 78% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO: 2)); a Homo sapiens mucolipin 1 protein (GenBank accession No.
  • NP 065394 (SEQ ID NO: 44); having 36% identity and 56% homology over 85% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO: 2)); a Mus musculus mucolipin 1 protein (GenBank accession No. NP_444407 (SEQ ID NO: 46); having 36% identity and 56% homology over 85% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO: 2)); a Homo sapiens mucolipin-3 protein (GenBank accession No.
  • NP_060768 (SEQ ID NO: 48); having 36% identity and 53% homology over 83% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO: 2)); a Homo sapiens mucolipidin protein (GenBank accession No. CAC07813 (SEQ ID NO: 50); having 40% identity and 59% homology over 54% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO: 2)); a Mus musculus mucolipin-1 protein (GenBank accession No.
  • AAL58667 (SEQ ID NO: 54); having 36% identity and 56% homology over 85 % of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO:2)); a Homo sapiens mucolipin 1 protein (GenBank accession No. AAH05149 (SEQ ID NO: 56); having 36% identity and 56% homology over 85% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO: 2)); a Homo sapiens mucolipin protein (GenBank accession No.
  • AAG00798 (SEQ ID NO: 58); having 36% identity and 56% homology over 85% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO: 2); a Homo sapiens mucolipin protein (GenBank accession No. AAG00797 (SEQ ID NO: 60); having 36% identity and 56% homology over 85% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO: 2)); a Mus musculus putative homolog to mucolipidin (GenBank accession No.
  • BAB31730 (SEQ ID NO: 62); having 37% identity and 58% homology over 78% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO: 2)); and a Homo sapiens mucolipidosis type IV protein (GenBank accession No. AAG10422 (SEQ ID NO: 64); having 36% identity and 56% homology over 85% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO: 2).
  • other proteins that may be used in the methods provided herein include, but are not limited to, proteins involved in intracellular calcium modulation that are substantially homologous to the above-listed proteins.
  • assays are conducted to detect an interaction between a molecule, such as, for example, a cellular component, and a protein involved in intracellular calcium modulation.
  • the interaction can be any direct or indirect physical, biochemical, chemical or other interaction between a molecule and a protein involved in intracellular calcium modulation such as, for example, those described herein.
  • one type of interaction that can occur between a candidate molecule involved in modulating intracellular calcium and a protein involved in intracellular calcium modulation is a binding interaction.
  • the protein involved in intracellular calcium modulation is contacted with isolated cellular components, cell medium, a cell and/or a cell extract and assayed for binding of a molecule to the protein involved in modulating intracellular calcium. Binding can be evaluated and detected using any of several methods known in the art for detecting binding of molecules to proteins, including, but not limited to, affinity chromatography (including Biacore and Ciphergen technologies), immunoprecipitation, ELISA assays, far-western blotting and other methods. Immunoassays to detect binding of molecules to a protein involved in intracellular calcium modulation can utilize antibodies (e.g., monoclonal and polyclonal) prepared against the protein or portions thereof. Methods of generating and testing antibodies against proteins are well known in the art.
  • a molecule that has bound to the protein involved in intracellular calcium modulation can be characterized and identified using methods that are also well known in the art, including, for example, HPLC, FPLC, amino acid sequencing if the molecule is a protein, and cloning of nucleic acid encoding a molecule that is a protein.
  • a molecule Once a molecule is found to interact with a protein involved in modulating intracellular calcium, it can be further evaluated to assess its intracellular calcium-modulating properties. For example, a cell comprising the interacting molecule and the protein involved in modulating intracellular calcium can be used for such evaluations. The interaction between the molecule and the protein involved in intracellular calcium modulation can be disrupted or altered in the cell and then intracellular calcium can be monitored to determine if intracellular calcium is altered by the alteration in the interaction. Intracellular calcium can be assessed using any methods known in the art or described herein. In another process for assessing the intracellular calcium-modulating properties of an interacting molecule, the amount of the molecule in such a cell can also be altered, for example, increased, decreased or the molecule can be eliminated in the cell.
  • One method for altering the amount of the molecule in the cell, if the molecule is a protein produced by the cell, is to alter the expression of the protein in the cell. This can be accomplished using a variety of methods, including methods described herein, such as for example, RNA interference, antisense RNA methods, gene knock-out procedures and gene insertion/over- expression processes.
  • F. Methods of Modulating Intracellular Calcium Provided herein are methods for modulating intracellular calcium.
  • Modulation of intracellular calcium can be any alteration or adjustment in intracellular calcium including but not limited to alteration of calcium concentration or level in the cytoplasm and/or intracellular calcium storage organelles, e.g., endoplasmic reticulum, alteration in the movement of calcium into, out of and within a cell, alteration in the location of calcium within a cell, and alteration of the kinetics, or other properties, of calcium fluxes into, out of and within cells.
  • intracellular calcium modulation can involve an alteration in basal or resting cytosolic calcium levels and/or store- operated calcium entry into the cell.
  • modulation of intracellular calcium can involve an alteration in receptor-mediated ion (e.g., calcium) movement, second messenger-operated ion (e.g. , calcium) movement, calcium influx into or efflux out of a cell, and/or ion (e.g., calcium) uptake into or release from intracellular compartments, including, for example, endosomes and lysosomes.
  • receptor-mediated ion e.g., calcium
  • second messenger-operated ion e.g. , calcium
  • calcium influx into or efflux out of a cell e.g., calcium
  • ion e.g., calcium
  • Methods of modulating intracellular calcium include a step of modulating the level, functioning and/or activity of one or more proteins involved in modulating intracellular calcium.
  • the methods may include a step of contacting a cell with an agent that modulates the level, functioning and/or activity of one or more proteins involved in modulating intracellular calcium.
  • the protein involved in modulating intracellular calcium can be, for example, an ion transport protein, calcium-binding protein or a protein that regulates an ion transport protein.
  • the protein is homologous to a protein encoded by a Drosophila gene that, when altered in its expression in a Drosophila cell, results in altered store-operated calcium entry into the cell.
  • An alteration of store-operated ion flux into a cell can be a complete or nearly complete elimination of the activity, a reduction of the activity, an alteration in properties or characteristics (e.g., current properties or sensitivities) of the activity or an increase in the activity relative to the activity in a control cell (e.g., a cell such as a Drosophila cell) that has not been altered in its store-operated ion flux activity.
  • a control cell e.g., a cell such as a Drosophila cell
  • an alteration in gene expression may be complete or nearly complete elimination of the expression of a gene, a reduction in the expression of a gene, an increase in the expression of a gene, or an alteration in the protein encoded by the gene (such as truncation or other alteration that effectively renders the protein nonfunctional or provides for aberrant functioning of the protein) relative to the expression of the gene in a cell that has not been altered in its expression of the gene.
  • the protein is homologous to a protein encoded by a Drosophila gene that, when altered in its expression in a Drosophila cell, results in altered intracellular calcium in the cell.
  • An alteration in intracellular calcium can be any alteration in calcium level, movement, location, or other calcium alteration, in a cell.
  • an alteration in intracellular calcium can be an alteration in the calcium level within an intracellular organelle or calcium storage compartment or an alteration in basal or resting cytosolic calcium levels.
  • An alteration of intracellular calcium can be any change in intracellular calcium compared to intracellular calcium in a control cell (e.g., a Drosophila cell that does not have altered expression of the gene).
  • an alteration of intracellular calcium can be, for example, an increase or decrease in basal or resting cytosolic calcium levels compared to control levels (e.g., levels in a Drosophila cell that does not have altered expression of the gene).
  • Assessment of intracellular calcium can be conducted in a number of ways, such as by methods described herein or known in the art. For example, assessment of basal or resting calcium levels can be conducted as described in Example 3 and by using any methods known in the art.
  • An alteration in basal or resting cytosolic calcium can also be effected in a number of ways, including, but not limited to, alterations in calcium flux across the plasma membrane or membranes of intracellular organelles such as endocytic organelles.
  • the protein is homologous to a protein encoded by a Drosophila gene that, when altered in its expression in a Drosophila cell, results in altered store-operated calcium entry into the cell and altered basal or resting cytosolic calcium levels.
  • the protein involved in modulating intracellular calcium has an amino acid sequence that is at least about 20%, or at least about 25%, or at least about 30%, or least about 35%, or at least about 40%, or at least about 45%, or at least about 50%, or at least about 52%, or at least about 55%, or at least about 60%, or at least about 65%, or at least about 70%, or at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 95% or more homologous to the protein encoded by the coding sequence of a Drosophila gene that, when altered in its expression, results in altered store-operated calcium entry into the cell and/or altered intracellular calcium.
  • the particular homology can depend on the particular protein, e.g., species, that is homologous to the Drosophila gene, the extent of the encoded Drosophila protein to which the particular protein is homologous, and can also depend on the particular Drosophila gene that, when altered in its expression in a Drosophila cell, results in altered store-operated calcium entry into the cell and/or altered intracellular calcium.
  • the protein is at least 52% or more homologous to the protein encoded by the coding sequence of the Drosophila gene.
  • Such exemplary proteins may be homologous to the specified Drosophila protein over at least about 25%, or at least about 30%, or at least about 35%, or at least about 40%, or at least about 45%, or at least about 50%, or at least about 54%, or least about 55%, or at least about 60%, or at least about 65%, or at least about 70%, or at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 95% or more of the protein encoded by the coding sequence of the Drosophila gene.
  • the protein is homologous to the Drosophila protein over at least about 54% or more of the protein encoded by the coding sequence of the Drosophila gene.
  • the one or more proteins involved in intracellular calcium modulation is at least about 52% homologous over at least about 54% of the protein encoded by the coding sequence of the Drosophila gene that, when altered in its expression, results in altered store-operated calcium entry into the cell and/or altered intracellular calcium.
  • the cell is contacted with an agent that modulates the level and/or activity of a protein homologous to a protein encoded by the coding sequence of Drosophila gene CG8743 (Genbank Accession No. AAF491 18; gi7293750; see also SEQ ID NO. 1 for gene coding sequence and SEQ ID NO. 2 for amino acid sequence).
  • Exemplary proteins include proteins that have an amino acid sequence that is at least about 20%, or at least about 25%, or at least about 30%, or least about 35%, or at least about 40%, or at least about 45%, or at least about 50%, or at least about 52%, or at least about 55%, or at least about 60%, or at least about 65%, or at least about 70%, or at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 95% or more homologous to an amino sequence of the protein encoded by the coding sequence of at least Drosophila gene CG8743.
  • the particular homology can depend on the particular protein, e.g., species, that is homologous to the protein encoded by Drosophila gene CG8743 and the extent of the protein encoded by Drosophila gene CG8743 to which the particular protein is homologous.
  • the protein is at least 52% or more homologous to the protein encoded by the coding sequence of at least Drosophila gene CG8743.
  • Such exemplary proteins may be homologous to the specified Drosophila protein over at least about 25%, or at least about 30%, or at least about 35%, or at least about 40%, or at least about 45%, or at least about 50%, or at least about 54%, or least about 55%, or at least about 60%, or at least about 65%, or at least about 70%, or at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 95% or more of the protein encoded by the coding sequence of Drosophila gene CG8743.
  • the protein is homologous to the Drosophila protein over at least about 54% or more of the protein encoded by the coding sequence of CG8743.
  • the protein has an amino acid sequence that is at least about 52% homologous over at least about 54% of the protein encoded by the coding sequence of at least Drosophila gene CG8743.
  • Proteins homologous to a protein encoded by the coding sequence of Drosophila gene CG8743 include, but are not limited to, the following: Homo sapiens mucolipin-3 protein (GenBank accession No. AAL84622 (SEQ ID NO: 4); having 41 % identity and 61 % homology over 83% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO:2)); a Mus musculus mucolipin-3 (GenBank accession No.
  • AAL84623 (SEQ ID NO: 6); having 40% identity and 60% homology over 86% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO: 2)); a Mus musculus unnamed, putative mucolipidin (Genbank accession No. BAB29372 (SEQ ID NO: 8); having 39% identity and 60% homology over 78% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO: 2)); a C. elegans hypothetical protein R13A5.1 a (GenBank accession No.
  • AAL38964 (SEQ ID NO: 10); having 37% identity and 59% homology over 84% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO: 2)); a C. elegans hypothetical protein R13A5.1 a.p (GenBank accession No. NP 498664 (SEQ ID NO: 1 2); having 37% identity and 59% homology over 84% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO: 2)); a C. elegans hypothetical protein R13A5.1 b.p (GenBank accession No.
  • NP_498665 (SEQ ID NO: 14)); having 37% identity and 59% homology over 84% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO: 2)); a Homo sapiens mucolipidin protein (GenBank accession No. CAC08215 (SEQ ID NO: 1 6)); having 38% identity and 58% homology over 85% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO:2)) ; a C. elegans CUP-5L protein (GenBank accession No.
  • AAL89754 (SEQ ID NO: 1 8); having 37% identity and 59% homology over 84% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO: 2)); a C. elegans CUP-5 protein (GenBank accession No. AAK19624 (SEQ ID NO: 20); having 37% identity and 59% homology over 84% of the protein encoded by the coding sequence of Drosophila gene CG8743 SEQ ID NO: 2)); a C. elegans hypothetical protein R1 3A5.1 b (GenBank accession No.
  • AAL38965 (SEQ ID NO: 22); having 36% identity and 58% homology over 85 % of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO: 2)); a Mus musculus unnamed protein similar to mucolipin (GenBank accession No. AAH05651 (SEQ ID NO: 24); having 38% identity and 58% homology over
  • BAA91 951 (SEQ ID NO: 28); having 38% identity and 55% homology over 83% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO: 2)); a Homo sapiens mucolipidin protein (GenBank accession No. CAC07813 (SEQ ID NO: 30); having 43% identity and 63% homology over 54% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO: 2)); a Homo sapiens hypothetical protein (GenBank accession No.
  • XP_088847 (SEQ ID NO: 32); having 35% identity and 52% homology over 77% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO: 2)); a Mus musculus mucolipin-3 protein (Genbank accession No. NP 598921 (SEQ ID NO: 34); having 38% identity and 58% homology over
  • AAM1 5597 (SEQ ID NO: 38); having 35% identity and 56% homology over 84% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO: 2)); a Mus musculus mucolipin-2 protein (Genbank accession No. AAM28596 (SEQ ID NO: 40); having 37% identity and 58% homology over 78% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO: 2)); a Mus musculus RIKEN cDNA 3300002C04 protein (Genbank accession No.
  • NP_080932 (SEQ ID NO: 42); having 37% identity and 58% homology over 78% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO: 2)); a Homo sapiens mucolipin 1 protein (GenBank accession No. NP_065394 (SEQ ID NO 44; having 36% identity and 56% homology over 85% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO: 2)); a Mus musculus mucolipin 1 protein (GenBank accession No.
  • NP_444407 SEQ ID NO: 46; having 36% identity and 56% homology over 85% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO:2)); a Homo sapiens mucolipin-3 protein (GenBank accession No. NP 060768 (SEQ ID NO: 48); having 36% identity and 53% homology over 83% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO 2)); a Homo sapiens mucolipidin protein (GenBank accession No.
  • CAC07813 (SEQ ID NO: 50); having 40% identity and 59% homology over 54% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO: 2)); a C. elegans CUP-5 protein (GenBank accession No. AAM15596 (SEQ ID NO: 52); having 35% identity and 56% homology over 84% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO: 2)); a Mus musculus mucolipin-1 protein (GenBank accession No.
  • AAL58667 (SEQ ID NO: 54); having 36% identity and 56% homology over 85% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO: 2)); a Homo sapiens mucolipin 1 protein (GenBank accession No. AAH05149 (SEQ ID NO: 56); having 36% identity and 56% homology over 85% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO: 2)); a Homo sapiens mucolipin protein (GenBank accession No.
  • AAG00798 (SEQ ID NO: 58); having 36% identity and 56% homology over 85% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO: 2)); a Homo sapiens mucolipin protein (GenBank accession No. AAG00797 (SEQ ID NO: 60); having 36% identity and 56% homology over 85% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO: 2)); a Mus musculus putative homolog to mucolipidin (GenBank accession No.
  • BAB31730 (SEQ ID NO: 62); having 37% identity and 58% homology over 78% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO: 2)); and a Homo sapiens mucolipidosis type IV protein (GenBank accession No. AAG10422 (SEQ ID NO: 64); having 36% identity and 56% homology over 85% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO: 2)).
  • other proteins that may be used in the methods provided herein include, but are not limited to, proteins involved in intracellular calcium modulation that are substantially homologous to the above-listed proteins.
  • a cell is contacted with an agent that modulates the level and/or activity of mucolipidin (also referred to as mucolipin) or a mucolipidin-like protein.
  • mucolipidin also referred to as mucolipin
  • exemplary mucolipidin or mucolipidin-like proteins include the following: Homo sapiens mucolipin-3 protein (GenBank accession No. AAL84622 (SEQ ID NO: 4); having 41 % identity and 61 % homology over 83% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO: 2)); a Mus musculus mucolipin-3 (GenBank accession No.
  • AAL84623 (SEQ ID NO: 6); having 40% identity and 60% homology over 86% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO: 2)); a Mus musculus unnamed, putative mucolipidin (GenBank accession No. BAB29372 (SEQ ID NO: 8); having 39% identity and 60% homology over 78% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO: 2)); a Homo sapiens mucolipidin protein (GenBank accession No.
  • CAC08215 (SEQ ID NO: 16); having 38% identity and 58% homology over 85% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO: 2)); a Mus musculus unnamed protein similar to mucolipin 1 protein (GenBank accession No. AAH05651 (SEQ ID NO: 24); having 38% identity and 58% homology over 85% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO: 2)); a Homo sapiens mucolipin 1 protein (GenBank accession No.
  • AAG42242 (SEQ ID NO: 26); having 37% identity and 56% homology over 87% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO: 2)); a Homo sapiens mucolipidin protein (GenBank accession No. CAC07813 (SEQ ID NO: 30); having 43% identity and 63% homology over 54% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO: 2)); a Mus musculus mucolipin-3 protein (GenBank accession No.
  • NP 598921 (SEQ ID NO: 34); having 38% identity and 58% homology over 86% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO: 2)); a Mus musculus mucolipin-2 protein (GenBank accession No. AAM28596 (SEQ ID NO: 40); having 37% identity and 58% homology over 78% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO: 2)); a Homo sapiens mucolipin 1 protein (GenBank accession No.
  • NP_065394 (SEQ ID NO: 44); having 36% identity and 56% homology over 85% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO: 2)); a Mus musculus mucolipin 1 protein (GenBank accession No. NP_444407 (SEQ ID NO: 46); having 36% identity and 56% homology over 85% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO: 2)); a Homo sapiens mucolipin-3 protein (GenBank accession No.
  • NP_ 060768 SEQ ID NO: 48; having 36% identity and 53% homology over 83% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO: 2)); a Homo sapiens mucolipidin protein (GenBank accession No. CAC07813 (SEQ ID NO: 50); having 40% identity and 59% homology over 54% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO: 2)); a Mus musculus mucolipin-1 protein (GenBank accession No.
  • AAL58667 (SEQ ID NO: 54); having 36% identity and 56% homology over 85% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO:2)); a Homo sapiens mucolipin 1 protein (GenBank accession No. AAH05149 (SEQ ID NO: 56); having 36% identity and 56% homology over 85% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO: 2)); a Homo sapiens mucolipin protein (GenBank accession No.
  • AAG00798 (SEQ ID NO: 58); having 36% identity and 56% homology over 85% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO: 2); a Homo sapiens mucolipin protein (GenBank accession No. AAG00797 (SEQ ID NO: 60); having 36% identity and 56% homology over 85% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO: 2)); a Mus musculus putative homolog to mucolipidin (GenBank accession No.
  • BAB31730 (SEQ ID NO: 62); having 37% identity and 58% homology over 78% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO: 2)); and a Homo sapiens mucolipidosis type IV protein (GenBank accession No. AAG10422 (SEQ ID NO: 64); having 36% identity and 56% homology over 85% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO: 2).
  • other proteins that may be used in the methods provided herein include, but are not limited to, proteins involved in intracellular calcium modulation that are substantially homologous to the above-listed proteins.
  • mucolipidin or mucolipidin-like proteins include the following: Homo sapiens mucolipin-3 protein (GenBank accession No. AAL84622; having 41 % identity and 61 % homology over 83% of the protein encoded by the coding sequence of Drosophila gene CG8743); a Mus musculus mucolipin-3 (GenBank accession No. AAL84623; having 40% identity and 60% homology over 86% of the protein encoded by the coding sequence of Drosophila gene CG8743); a Mus musculus unnamed, putative mucolipidin (GenBank accession No.
  • BAB29372 having 39% identity and 60% homology over 78% of the protein encoded by the coding sequence of Drosophila gene CG8743); a Homo sapiens mucolipidin protein (GenBank accession No. CAC0821 5; having 38% identity and 58% homology over 85% of the protein encoded by the coding sequence of Drosophila gene CG8743); a Mus musculus unnamed protein similar to mucolipin 1 (GenBank accession No. AAH05651 ; having 38% identity and 58% homology over 85% of the protein encoded by the coding sequence of Drosophila gene CG8743); a Homo sapiens mucolipin 1 protein (GenBank accession No.
  • AAG42242 having 37% identity and 56% homology over 87% of the protein encoded by the coding sequence of Drosophila gene CG8743); a Homo sapiens mucolipidin protein (GenBank accession No. CAC07813; having 43% identity and 63% homology over 54% of the protein encoded by the coding sequence of Drosophila gene CG8743); a Mus musculus mucolipin-3 protein (GenBank accession No. NP_598921 ; having 38%> identity and 58% homology over 86% of the protein encoded by the coding sequence of Drosophila gene CG8743); a Mus musculus mucolipin-2 protein (GenBank accession No.
  • AAM28596 having 37% identity and 58% homology over 78% of the protein encoded by the coding sequence of Drosophila gene CG8743); a Homo sapiens mucolipin 1 protein (GenBank accession No. NP 065394; having 36% identity and 56% homology over 85% of the protein encoded by the coding sequence of Drosophila gene CG8743); a Mus musculus mucolipin 1 protein (GenBank accession No. NP_444407; having 36% identity and 56% homology over 85% of the protein encoded by the coding sequence of Drosophila gene CG8743); a Homo sapiens mucolipin-3 protein (GenBank accession No.
  • NP 060768 having 36% identity and 53% homology over 83% of the protein encoded by the coding sequence of Drosophila gene CG8743
  • a Homo sapiens mucolipidin protein GenBank accession No. CAC07813; having 40% identity and 59% homology over 54% of the protein encoded by the coding sequence of Drosophila gene CG8743
  • a Mus musculus mucolipin-1 protein GenBank accession No. AAL58667; having 36% identity and 56% homology over 85% of the protein encoded by the coding sequence of Drosophila gene CG8743
  • a Homo sapiens mucolipin 1 protein GenBank accession No.
  • AAH05149 having 36% identity and 56% homology over 85% of the protein encoded by the coding sequence of Drosophila gene CG8743
  • a Homo sapiens mucolipin protein GenBank accession No. AAG00798; having 36% identity and 56% homology over 85% of the protein encoded by the coding sequence of Drosophila gene CG8743
  • a Homo sapiens mucolipin protein GenBank accession No. AAG00797; having 36% identity and 56% homology over 85% of the protein encoded by the coding sequence of Drosophila gene CG8743
  • a Mus musculus putative homolog to mucolipidin GenBank accession No.
  • BAB31730 having 37% identity and 58% homology over 78% of the protein encoded by the coding sequence of Drosophila gene CG8743); and a Homo sapiens mucolipidosis type IV protein (GenBank accession No. AAG10422; having 36% identity and 56% homology over 85% of the protein encoded by the coding sequence of Drosophila gene CG8743).
  • other proteins that may be used in the methods provided herein include, but are not limited to, proteins involved in intracellular calcium modulation that are substantially homologous to the above-listed proteins.
  • Agents that can be used in the methods of modulating intracellular calcium can modulate the level, functioning and/or activity of a protein involved in modulating intracellular calcium, such as those described herein or identified using methods described herein.
  • Agents that can modulate the level of the protein include, but are not limited to, substances that affect the expression of nucleic acid encoding the protein in a cell.
  • the agent can be a nucleic acid, e.g., genomic DNA, cDNA or expression vector, that is introduced into the cell for expression of the protein therein, thereby increasing the level of the protein in the cell.
  • the agent can be a nucleic acid that is introduced into the cell to increase or decrease the amount of protein produced in the cell.
  • the agent can be a cDNA or expression vector that provides for expression of the protein in the cell or serves to disrupt the already present DNA encoding in the protein such that the level of the protein is decreased in the cell.
  • the agent can also be RNA, e.g., antisense or dsRNA, that results in reduction of the level of the protein in the cell.
  • Agents that can modulate the activity of the protein include, but are not limited to, substances that increase, decrease or otherwise alter the activity and/or functioning of the protein.
  • Activities of calcium-modulating proteins include, but are not limited to, calcium transport, calcium binding, and regulation of calcium-modulating proteins.
  • agents that modulate these activities, or any other activity involved in intracellular calcium modulation can be used in the methods provided herein.
  • antibodies or other proteins that specifically bind to the protein and modulate such activities can be agents used in the methods.
  • An antibody or other protein may, for instance, bind to a site of a regulatory protein and reduce or eliminate its binding to the protein it regulates, thereby reducing its regulatory activity.
  • Ions other than calcium or molecules, such as small organic molecules can also be agents that may, for example, reduce or increase calcium transport by an ion transport protein and thus can be agents used in the methods.
  • Methods of Identifying Agents for the Treatment of a Disease or Disorder Disease models are a valuable tool for the discovery and testing of treatment agents.
  • Such disease models may be cellular or organismal and may be produced by methods known to those of skill in the art and described herein.
  • Provided herein are models for diseases and disorders involving or characterized at least in part by calcium dyshomeostasis or alterations in calcium signalling. Also provided herein are methods of identifying candidate agents for the treatment of such diseases and disorders which utilize the models. 1.
  • Cell models for diseases and disorders involving or characterized at least in part by calcium dyshomeostasis or alterations in calcium signalling are also provided herein.
  • Cell models for the identification and testing of candidate agents for the treatment of diseases and disorders involving or characterized at least in part by calcium dyshomeostasis or alterations in calcium signalling are provided herein.
  • the cell models can also be used in elucidating the mechanisms underlying calcium dyshomeostasis or altered calcium signalling in a cell as well as in dissecting processes involved in intracellular calcium regulation.
  • the cell model includes a recombinant cell that contains heterologous nucleic acid encoding one or more proteins involved in intracellular calcium modulation homologous to a protein encoded by a Drosophila gene that, when altered in its expression in a Drosophila cell, results in altered store-operated calcium entry into the cell.
  • An alteration of store- operated ion flux into a cell can be a complete or nearly complete elimination of the activity, a reduction of the activity, an alteration in properties or characteristics (e.g., current properties or sensitivities) of the activity or an increase in the activity relative to the activity in a control cell (e.g., a cell such as a Drosophila cell) that has not been altered in its store-operated ion flux activity.
  • a control cell e.g., a cell such as a Drosophila cell
  • an alteration in gene expression may be complete or nearly complete elimination of the expression of a gene, a reduction in the expression of a gene, an increase in the expression of a gene, or an alteration in the protein encoded by the gene (such as truncation or other alteration that effectively renders the protein nonfunctional or provides for aberrant functioning of the protein) relative to the expression of the gene in a cell that has not been altered in its expression of the gene.
  • the cell model includes a recombinant cell that contains heterologous nucleic acid encoding one or more proteins involved in intracellular calcium modulation homologous to a protein encoded by a Drosophila gene that, when altered in its expression in a Drosophila cell, results in altered intracellular calcium in the cell.
  • An alteration in intracellular calcium can be any alteration in calcium level, movement, location, or other calcium alteration, in a cell.
  • an alteration in intracellular calcium can be an alteration in the calcium level within an intracellular organelle or calcium storage compartment or an alteration in basal or resting cytosolic calcium levels.
  • An alteration of intracellular calcium can be any change in intracellular calcium compared to intracellular calcium in a control cell (e.g., a Drosophila cell that does not have altered expression of the gene).
  • an alteration of intracellular calcium can be, for example, an increase or decrease in basal or resting cytosolic calcium levels compared to control levels (e.g., levels in a Drosophila cell that does not have altered expression of the gene).
  • Assessment of intracellular calcium can be conducted in a number of ways, such as by methods described herein or known in the art. For example, assessment of basal or resting calcium levels can be conducted as described in Example 3 and by using any methods known in the art.
  • An alteration in basal or resting cytosolic calcium can also be effected in a number of ways, including, but not limited to, alterations in calcium flux across the plasma membrane or membranes of intracellular organelles such as endocytic organelles.
  • the cell model includes a recombinant cell that contains heterologous nucleic acid encoding one or more proteins involved in intracellular calcium modulation homologous to a protein encoded by a Drosophila gene that, when altered in its expression in a Drosophila cell, results in altered store-operated calcium entry into the cell and altered basal or resting cytosolic calcium levels.
  • the recombinant cell contains heterologous nucleic acid encoding one or more proteins that has an amino acid sequence that is at least about 20%, or at least about 25%, or at least about 30%, or least about 35%, or at least about 40%, or at least about 45%, or at least about 50%, or at least about 52%, or at least about 55%, or at least about 60%, or at least about 65%, or at least about 70%, or at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 95% or more homologous to the protein encoded by the coding sequence of a Drosophila gene that, when altered in its expression, results in altered store-operated calcium entry into the cell and/or altered intracellular calcium.
  • the particular homology can depend on the particular protein, e.g., species, that is homologous to the Drosophila gene, the extent of the encoded Drosophila protein to which the particular protein is homologous, and can also depend on the particular Drosophila gene that, when altered in its expression in a Drosophila cell, results in altered store-operated calcium entry into the cell and/or altered intracellular calcium.
  • the protein is at least 52% or more homologous to the protein encoded by the coding sequence of the Drosophila gene.
  • Such exemplary proteins may be homologous to the specified Drosophila protein over at least about 25%, or at least about 30%, or at least about 35%, or at least about 40%, or at least about 45%, or at least about 50%, or at least about 54%, or least about 55%, or at least about 60%, or at least about 65%, or at least about 70%, or at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 95% or more of the protein encoded by the coding sequence of the Drosophila gene.
  • the protein is homologous to the Drosophila protein over at least about 54% or more of the protein encoded by the coding sequence of the Drosophila gene.
  • the one or more proteins encoded by the heterologous nucleic acid is at least about 52% homologous over at least about 54% of the protein encoded by the coding sequence of the Drosophila gene that, when altered in its expression, results in altered store-operated calcium entry into the cell and/or altered intracellular calcium.
  • the cell model includes a recombinant cell that contains heterologous nucleic acid encoding one or more proteins homologous to a protein encoded by the coding sequence of Drosophila gene CG8743 (Genbank Accession No. AAF491 1 8; gi7293750; see also SEQ ID NO. 1 for gene coding sequence and SEQ ID NO. 2 for amino acid sequence).
  • Exemplary proteins include proteins that have an amino acid sequence that is at at least about 20%, or at least about 25 %, or at least about 30%, or least about 35 %o, or at least about 40%, or at least about 45%, or at least about 50%, or at least about 52%, or at least about 55%, or at least about 60%, or at least about 65%, or at least about 70%, or at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 95% or more homologous to an amino acid sequence of the protein encoded by the coding sequence of at least Drosophila gene CG8743.
  • the particular homology can depend on the particular protein, e.g.
  • the protein is at least 52% or more homologous to the protein encoded by the coding sequence of at least Drosophila gene CG8743.
  • Such exemplary proteins may be homologous to the specified Drosophila protein over at least about 25%, or at least about 30%, or at least about 35%, or at least about 40%, or at least about 45%, or at least about 50%, or at least about 54%, or least about 55%, or at least about 60%, or at least about 65%, or at least about 70%, or at least about 75%, or at least about 80%, or at least about 85 %, or at least about 90%, or at least about 95% or more of the protein encoded by the coding sequence of Drosophila gene CG8743.
  • the protein is homologous to the Drosophila protein over at least about 54% or more of the protein encoded by the coding sequence of CG8743.
  • the protein has an amino acid sequence that is at least about 52% homologous over at least about 54% of the protein encoded by the coding sequence of at least Drosophila gene CG8743.
  • Proteins homologous to a protein encoded by the coding sequence of Drosophila gene CG8743 include, but are not limited to, the following: Homo sapiens mucolipin-3 protein (GenBank accession No. AAL84622 (SEQ ID NO: 4); having 41 % identity and 61 % homology over 83% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO:2)); a Mus musculus mucolipin-3 (GenBank accession No.
  • AAL84623 (SEQ ID NO: 6); having 40% identity and 60% homology over 86%) of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO: 2)); a Mus musculus unnamed, putative mucolipidin (Genbank accession No. BAB29372 (SEQ ID NO: 8); having 39% identity and 60%> homology over 78% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO: 2)); a C. elegans hypothetical protein R13A5.1 a (GenBank accession No. AAL38964 (SEQ ID NO: 10); having 37% identity and 59% homology over 84% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO: 2)); a C. elegans hypothetical protein
  • R1 3A5.1 a.p GenBank accession No. NP_498664 (SEQ ID NO: 1 2); having 37% identity and 59% homology over 84% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO: 2)); a C. elegans hypothetical protein R13A5.1 b.p (GenBank accession No. NP 498665 (SEQ ID NO: 14)); having 37% identity and 59% homology over 84% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO: 2)); a Homo sapiens mucolipidin protein (GenBank accession No.
  • CAC0821 5 (SEQ ID NO: 1 6)); having 38% identity and 58% homology over 85% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO:2)) ; a C. elegans CUP-5L protein (GenBank accession No. AAL89754 (SEQ ID NO: 1 8); having 37% identity and 59% homology over 84% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO: 2)); a C. elegans CUP-5 protein (GenBank accession No.
  • AAK1 9624 (SEQ ID NO: 20); having 37 % identity and 59% homology over 84% of the protein encoded by the coding sequence of Drosophila gene CG8743 SEQ ID NO: 2)); a C. elegans hypothetical protein R1 3A5.1 b (GenBank accession No. AAL38965 (SEQ ID NO: 22); having 36% identity and 58% homology over 85% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO: 2)); a Mus musculus unnamed protein similar to mucolipin (GenBank accession No.
  • AAH05651 (SEQ ID NO: 24); having 38% identity and 58% homology over 85% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO: 2)); a Homo sapiens mucolipin 1 protein (GenBank accession No. AAG42242 (SEQ ID NO: 26); having 37% identity and 56% homology over 87% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO: 2)); a Homo sapiens unnamed protein product (GenBank accession No.
  • BAA91951 (SEQ ID NO: 28); having 38% identity and 55% homology over 83% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO: 2)); a Homo sapiens mucolipidin protein (GenBank accession No. CAC07813 (SEQ ID NO: 30); having 43% identity and 63% homology over 54% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO: 2)); a Homo sapiens hypothetical protein (GenBank accession No. XP 088847 (SEQ ID NO: 32); having 35% identity and 52% homology over
  • AAM1 5598 (SEQ ID NO: 36); having 35% identity and 56% homology over 84% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO: 2)); a C. elegans CUP-5 protein (Genbank accession No. AAM1 5597 (SEQ ID NO: 38); having 35% identity and 56% homology over 84% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO: 2)); a Mus musculus mucolipin-2 protein (Genbank accession No.
  • AAM28596 (SEQ ID NO: 40); having 37% identity and 58% homology over 78% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO: 2)); a Mus musculus RIKEN cDNA 3300002C04 protein (Genbank accession No. NP 080932 (SEQ ID NO: 42); having 37% identity and 58% homology over 78% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO: 2)); a Homo sapiens mucolipin 1 protein (GenBank accession No.
  • NP 065394 (SEQ ID NO 44; having 36% identity and 56% homology over 85% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO: 2)); a Mus musculus mucolipin 1 protein (GenBank accession No. NP_444407 (SEQ ID NO: 46); having 36% identity and 56% homology over 85% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO:2)); a Homo sapiens mucolipin-3 protein (GenBank accession No.
  • NP 060768 SEQ ID NO: 48; having 36% identity and 53% homology over 83% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO 2)); a Homo sapiens mucolipidin protein (GenBank accession No. CAC07813 (SEQ ID NO: 50); having 40% identity and 59% homology over 54% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO: 2)); a C. elegans CUP-5 protein (GenBank accession No.
  • AAM1 5596 (SEQ ID NO: 52); having 35% identity and 56% homology over 84% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO: 2)); a Mus musculus mucolipin-1 protein (GenBank accession No. AAL58667 (SEQ ID NO: 54); having 36% identity and 56% homology over 85% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO: 2)); a Homo sapiens mucolipin 1 protein (GenBank accession No.
  • AAH05149 (SEQ ID NO: 56); having 36% identity and 56% homology over 85% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO: 2)); a Homo sapiens mucolipin protein (GenBank accession No. AAG00798 (SEQ ID NO: 58); having 36% identity and 56% homology over 85% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO: 2)); a Homo sapiens mucolipin protein (GenBank accession No.
  • AAG00797 (SEQ ID NO: 60); having 36% identity and 56% homology over 85% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO: 2)); a Mus musculus putative homolog to mucolipidin (GenBank accession No. BAB31730 (SEQ ID NO: 62); having 37% identity and 58% homology over 78% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO: 2)); and a Homo sapiens mucolipidosis type IV protein (GenBank accession No.
  • the cell model includes a recombinant cell that contains heterologous nucleic acid encoding one or more mucolipidin (also referred to as mucolipin) or mucolipidin-like proteins.
  • mucolipidin also referred to as mucolipin
  • mucolipidin-like proteins include the following: Homo sapiens mucolipin-3 protein (GenBank accession No.
  • AAL84622 (SEQ ID NO: 4); having 41 % identity and 61 % homology over 83% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO: 2)); a Mus musculus mucolipin-3 (GenBank accession No. AAL84623 (SEQ ID NO: 6); having 40% identity and 60% homology over 86% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO: 2)); a Mus musculus unnamed, putative mucolipidin (GenBank accession No.
  • BAB29372 (SEQ ID NO: 8); having 39% identity and 60% homology over 78% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO: 2)); a Homo sapiens mucolipidin protein (GenBank accession No. CAC0821 5 (SEQ ID NO: 1 6); having 38% identity and 58% homology over 85 % of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO: 2)); a Mus musculus unnamed protein similar to mucolipin 1 protein (GenBank accession No.
  • AAH05651 (SEQ ID NO: 24); having 38% identity and 58% homology over 85% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO: 2)); a Homo sapiens mucolipin 1 protein (GenBank accession No. AAG42242 (SEQ ID NO: 26); having 37% identity and 56% homology over
  • NP 598921 (SEQ ID NO: 34); having 38% identity and 58%) homology over 86% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO: 2)); a Mus musculus mucolipin-2 protein (GenBank accession No. AAM28596 (SEQ ID NO: 40); having 37% identity and 58% homology over 78% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO: 2)); a Homo sapiens mucolipin 1 protein (GenBank accession No. NP 065394 (SEQ ID NO: 44); having 36% identity and 56% homology over
  • NP 060768 SEQ ID NO: 48; having 36% identity and 53% homology over 83% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO: 2)); a Homo sapiens mucolipidin protein (GenBank accession No. CAC07813 (SEQ ID NO: 50); having 40%) identity and 59% homology over 54% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO: 2)); a Mus musculus mucolipin-1 protein (GenBank accession No.
  • AAL58667 (SEQ ID NO: 54); having 36% identity and 56% homology over 85% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO:2)); a Homo sapiens mucolipin 1 protein (GenBank accession No. AAH05149 (SEQ ID NO: 56); having 36% identity and 56% homology over
  • AAG00797 (SEQ ID NO: 60); having 36% identity and 56% homology over 85% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO: 2)); a Mus musculus putative homolog to mucolipidin (GenBank accession No. BAB31730 (SEQ ID NO: 62); having 37% identity and 58% homology over 78% of the protein encoded by the coding sequence of
  • Drosophila gene CG8743 (SEQ ID NO: 2)); and a Homo sapiens mucolipidosis type IV protein (GenBank accession No. AAG 10422 (SEQ ID NO: 64); having 36% identity and 56% homology over 85% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO: 2).
  • other proteins that may be used in the methods provided herein include, but are not limited to, proteins involved in intracellular calcium modulation that are substantially homologous to the above-listed proteins.
  • the cell model includes a recombinant cell that contains heterologous nucleic acid encoding a polymorphic or mutant form of one or more of the above-identified proteins.
  • a polymorphic or mutant protein can be one that has an altered activity or function or one that has no activity or is non-functional, particularly relative to a wild-type or predominant form of the protein.
  • cell models provided herein for the identification and testing of candidate agents for the treatment of diseases and disorders involving or characterized by calcium dyshomeostasis or altered calcium regulation can include a recombinant cell in which expression of a protein involved in intracellular calcium modulation as identified above, which is either endogenously expressed in the cell or which is expressed in the cell from a heterologous nucleic acid, has been altered or eliminated, such as by replacing or modifying the promoter region or other regulatory region driving expression of nucleic acid encoding the protein.
  • Such a cell can be produced, using methods known in the art and described herein, by introduction into a cell of heterologous nucleic acid that either targets and alters DNA regulatory sequences associated with an endogenous gene or that links DNA encoding the protein to a particular expression regulation sequence(s).
  • Cell models including a recombinant cell in which expression of an endogenous protein involved in intracellular calcium modulation as identified above has been reduced or eliminated can be produced by disruption or elimination (e.g., through gene knock-out, antisense RNA or RNA interference methods) of the gene or RNA encoding the protein in the cell.
  • the cell model includes a recombinant cell that expresses one or more proteins involved in intracellular calcium regulation, such as identified above, as heterologous protein(s).
  • Such cells may overexpress or mis-express the heterologous protein(s).
  • a recombinant cell may be one that endogenously expresses the protein(s) and also has been transfected with additional copies of nucleic acid encoding the protein(s).
  • the host cell used in the generating the recombinant cell may be one that endogenously expresses little to none of the protein(s) of interest. Any cell may be used in generating the cell models.
  • the cell is a neuronal, nervous system- or tissue-derived cell or a brain cell.
  • cells from a known cell line can be used, such as from neuroblastoma SH-SY5Y cells, pheochromocytoma PC12 cells, neuroblastoma SK-N-BE(2)C cells, human SK-N-MC neuroblastoma cells, SMS-KCNR cells, human LAN-5 neuroblastoma cells, human GI-CA-N neuroblastoma cells, human GOTO neuroblastoma cells, mouse Neuro 2a (N2A) neuroblastoma cells and/or human IMR 32 neuroblastoma cells.
  • Cell lines include HEK 293, CHO (including CHO-K1 ), LTK " , N2A, H6, HGB, and Drosophlia S2 cells. The generation, maintenance and use of such cell lines is well known. 2. Animal models
  • Transgenic animal models and animals for the identification and testing of candidate agents for the treatment of diseases and disorders involving or characterized by calcium dyshomeostasis or altered calcium regulation are provided herein.
  • the animal models can also be used in elucidating the mechanisms underlying calcium dyshomeostasis in an organism as well as in dissecting processes involved in intracellular calcium regulation.
  • Transgenic animals include, but are not limited to, non-human animals, such as rodents (e.g., mice and rats), cows, chickens, pigs, goats, sheep, insects, Drosophila, nematodes, worms, C. elegans, monkeys, gorillas, and other primates.
  • the transgenic non-human animals are such that the expression of nucleic acid encoding one or more proteins as identified above with respect to cell models is altered or eliminated in at least some cells in the animal.
  • Alteration or elimination of expression of the protein(s) can be achieved in a number of ways. For example, expression can be altered by replacing or modifying the promoter region or other regulatory region of an endogenous gene encoding the protein(s) in the animal. Such an animal can be produced by promoting recombination between endogenous nucleic acid and an exogenous nucleic acid.
  • Increased expression of one or more of the proteins in a transgenic animal can be achieved by altering or replacing an endogenous promoter or by introducing additional copies of nucleic acid encoding the protein(s) into the animal.
  • Reduction or elimination of expression of one or more of the proteins can be achieved by disruption or "knockout" of endogenous genes in the animal.
  • such an animal can initially be produced by promoting homologous recombination between a gene of interest in its chromosome and the corresponding exogenous gene of interest that has been rendered biologically inactive (typically by insertion of a heterologous sequence, e.g., an antibiotic resistance gene).
  • a heterologous sequence e.g., an antibiotic resistance gene.
  • it is possible to reduce or eliminate expression of a gene encoding a protein by introduction of double- stranded RNA that contains sequence identical or complementary to at least a portion of the sequence of the gene of interest into the animal.
  • Transgenic animals also include animals containing nucleic acids encoding mutant or polymorphic forms of the one or more proteins. Such animals can be prepared by "knock-in” methods in which the endogenous form (e.g., "normal") of a gene encoding the protein(s) is replaced by a variant, such a mutant, or other form. It is also possible to replace one species', such as a rodent's, endogenous gene with a gene from another species, such as from a human. Transgenic animals also can be produced by non-homologous recombination into other sites in a chromosome; including animals that have a plurality of integration events.
  • nucleic acids into cells for generation of transgenic animals can be conducted using any known method of nucleic acid delivery, including, but not limited to, microinjection, lipofection and other modes of nucleic acid delivery.
  • the nucleic acids can be introduced into cells such as, for example, germline cells or somatic cells, such as an embryonic stem cell.
  • the nucleic acid can be introduced into a cell, such as an embryonic stem cell (ES), followed by injecting the ES cells into a blastocyst, and implanting the blastocyst into a foster mother, which is followed by the birth of a transgenic animal.
  • ES embryonic stem cell
  • Nuclear transfer methods in which nucleic acid being used to generate a transgenic animal is introduced into a nuclear donor cell containing a totipotent nucleus, followed by transfer of the donor nucleus into a recipient cell, e.g., an enucleated oocyte, which can be transferred to a recipient female for development into a transgenic animal.
  • homologous recombination is performed by transforming embryo-derived stem (ES) cells with a vector containing the insertionally inactivated gene of interest, such that homologous recombination occurs, followed by injecting the ES cells into a blastocyst, and implanting the blastocyst into a foster mother, followed by the birth of the chimeric animal ("knockout animal") in which a gene of interest has been inactivated (see Capecchi, Science 244:1 288-1292 (1989)).
  • the chimeric animal can be bred to produce homozygous knockout animals, which can then be used to produce additional knockout animals.
  • Knockout animals include, but are not limited to, mice, hamsters, sheep, pigs, cattle, and other non-human mammals.
  • the resulting animals can serve as models of diseases involving altered expression of a protein involved in intracellular calcium modulation.
  • Such knockout animals can be used to screen for candidate therapeutic agents or to test molecules that have already been identified as candidate therapeutic agents for the ability to treat or prevent such diseases or disorders.
  • Cell and animal models of diseases and disorders involving calcium dyshomeostasis described herein have a number of uses. For example, by evaluating the cellular or organismal phenotypes associated with the altered expression of proteins involved in intracellular calcium modulation in the cells/organisms and correlating such phenotypes with specific cellular molecules and processes, the disease/disorder models can be used in elucidating the mechanisms underlying calcium dyshomeostasis in a cell as well as in dissecting processes and pathways involved in intracellular calcium regulation.
  • the models can be used in screening agents and testing candidate agents for the treatment of diseases and disorders that involve calcium dyshomeostasis.
  • the model cells and/or organisms are contacted with a test or candidate agent.
  • intracellular calcium and/or calcium movement is evaluated. For example, resting cytosolic calcium levels and/or store-operated calcium entry in cells can be evaluated.
  • a variety of methods may be used for evaluating such activity and are described herein and known in the art.
  • a test agent that alters intracellular calcium and/or movement of calcium into, out of or within disease model cells that exhibit calcium dyshomeostasis is identified as an agent that modulates intracellular calcium and possibly as a candidate agent for the treatment of a disease involving calcium dyshomeostasis.
  • An agent that at least partially reverses or reduces or eliminates a disease trait or phenotype exhibited by a model cell or organism, or that tends to restore calcium homeostasis and/or modulates calcium signaling or movement to compensate for disease-associated abnormalities in intracellular calcium is identified as a candidate agent for the treatment of a disease involving calcium dyshomeostasis.
  • a candidate agent for use in treatment of a disease or disorder is thus one that ameliorates or eliminates the symptoms and/or manifestations of an inherited or acquired disease or disorder or one that cures the disease or disorder or at least partially restores the wild-type phenotype. This may include modulation of calcium homeostasis, resting cytosolic calcium levels, and/or store operated calcium entry in model cells and/or organisms.
  • Methods provided herein for identifying proteins involved in modulating intracellular calcium as well as agents that modulate intracellular calcium are useful in elucidating cellular processes for calcium homeostasis and signalling. Because the methods can identify proteins, which were not previously known to be involved in intracellular calcium modulation, as calcium-modulating proteins, they are also useful in the discovery of methods for treating diseases and disorders involving calcium dyshomeostasis or altered intracellular calcium regulation.
  • Such methods can include a step of administering to a subject having a disease or disorder involving calcium dyshomeostasis or altered intracellular calcium regulation, an agent that modulates the level and/or activity of a protein involved in modulation of intracellular calcium.
  • the agent being administered is one that modulates the level and/or activity of a protein that has an amino acid sequence that is homologous to a protein encoded by a Drosophila gene that, when altered in its expression in a Drosophila cell, results in altered store-operated calcium entry into the cell.
  • An alteration of store-operated ion flux into a cell can be a complete or nearly complete elimination of the activity, a reduction of the activity, an alteration in properties or characteristics of the activity or an increase in the activity relative to the activity in a cell that has not been altered in its store-operated ion flux activity.
  • an alteration in gene expression may be complete or nearly complete elimination of the expression of a gene, a reduction in the expression of a gene, an increase in the expression of a gene, or an alteration in the protein encoded by the gene (such as truncation or other alteration that effectively renders the protein nonfunctional or provides for aberrant functioning of the protein) relative to the expression of the gene in a cell that has not been altered in its expression of the gene.
  • the agent being administered is one that modulates the level and/or activity of a protein that has an amino acid sequence that is homologous to a protein encoded by a Drosophila gene that, when altered in its expression in a Drosophila cell, results in altered intracellular calcium in the cell.
  • An alteration in intracellular calcium can be any alteration in calcium level, movement, location, or other calcium alteration, in a cell.
  • an alteration in intracellular calcium can be an alteration in the calcium level within an intracellular organelle or calcium storage compartment or an alteration in basal or resting cytosolic calcium levels.
  • An alteration of intracellular calcium can be any change in intracellular calcium compared to intracellular calcium in a control cell (e.g., a Drosophila cell that does not have altered expression of the gene).
  • an alteration of intracellular calcium can be, for example, an increase or decrease in basal or resting cytosolic calcium levels compared to control levels (e.g., levels in a Drosophila cell that does not have altered expression of the gene).
  • Assessment of intracellular calcium can be conducted in a number of ways, such as by methods described herein or known in the art. For example, assessment of basal or resting calcium levels can be conducted as described in Example 3 and by using any methods known in the art.
  • An alteration in basal or resting cytosolic calcium can also be effected in a number of ways, including, but not limited to, alterations in calcium flux across the plasma membrane or membranes of intracellular organelles such as endocytic organelles.
  • the agent being administered is one that modulates the level and/or activity of a protein that has an amino acid sequence that is homologous to a protein encoded by a Drosophila gene that, when altered in its expression in a Drosophila cell, results in altered store-operated calcium entry into the cell and altered basal or resting cytosolic calcium levels.
  • the agent being administered is one that modulates the level and/or activity of a protein that has an amino acid sequence that is at least about 20%, or at least about 25%, or at least about 30%, or least about 35%, or at least about 40%, or at least about 45%, or at least about 50%, or at least about 52%, or at least about 55%, or at least about 60%, or at least about 65%, or at least about 70%, or at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 95% or more homologous to the protein encoded by the coding sequence of a Drosophila gene that, when altered in its expression, results in altered store-operated calcium entry into the cell and/or altered intracellular calcium.
  • the particular homology can depend on the particular protein, e.g., species, that is homologous to the Drosophila gene, the extent of the encoded Drosophila protein to which the particular protein is homologous, and can also depend on the particular Drosophila gene that, when altered in its expression in a Drosophila cell, results in altered store-operated calcium entry into the cell and/or altered intracellular calcium.
  • the protein is at least 52% or more homologous to the protein encoded by the coding sequence of the Drosophila gene.
  • Such exemplary proteins may be homologous to the specified Drosophila protein over at least about 25%, or at least about 30%, or at least about 35%, or at least about 40%, or at least about 45 %, or at least about 50%, or at least about 54%, or least about 55%, or at least about 60%, or at least about 65%, or at least about 70%, or at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 95% or more of the protein encoded by the coding sequence of the Drosophila gene.
  • the protein is homologous to the Drosophila protein over at least about 54% or more of the protein encoded by the coding sequence of the Drosophila gene.
  • the protein is at least about 52% homologous over at least about 54% of the protein encoded by the coding sequence of the Drosophila gene that, when altered in its expression, results in altered store-operated calcium entry into the cell and/or altered intracellular calcium.
  • the agent being administered is one that modulates the level and/or activity of a protein homologous to a protein encoded by the coding sequence of Drosophila gene CG8743 (Genbank Accession No. AAF491 1 8; gi7293750; see also SEQ ID NO. 1 for gene coding sequence and SEQ ID NO. 2 for amino acid sequence).
  • the agent administered to the subject is one that modulates the level and/or activity of a protein involved in modulation of intracellular calcium that has an amino acid sequence that is at least about 20%, or at least about 25 %, or at least about 30%, or least about 35 %, or at least about 40%, or at least about 45%, or at least about 50%, or at least about 52%, or at least about 55%, or at least about 60%, or at least about 65%, or at least about 70%, or at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 95% or more homologous to an amino acid sequence of the protein encoded by the coding sequence of at least Drosophila gene CG8743.
  • the particular homology can depend on the particular protein, e.g., species, that is homologous to the protein encoded by Drosophila gene CG8743 and the extent of the protein encoded by Drosophila gene CG8743 to which the particular protein is homologous.
  • the protein is at least 52% or more homologous to the protein encoded by the coding sequence of at least Drosophila gene CG8743.
  • Such exemplary proteins may be homologous to the specified Drosophila protein over at least about 25%, or at least about 30%, or at least about 35%, or at least about 40%, or at least about 45%, or at least about 50%, or at least about 54%, or least about 55%, or at least about 60%, or at least about 65%, or at least about 70%, or at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 95% or more of the protein encoded by the coding sequence of Drosophila gene CG8743.
  • the protein is homologous to the Drosophila protein over at least about 54% or more of the protein encoded by the coding sequence of CG8743.
  • the agent administered to the subject is one that modulates the level and/or activity of a protein involved in modulation of intracellular calcium that has an amino acid sequence that is at least about 52% homologous over at least about 54% of the protein encoded by the coding sequence of at least Drosophila gene CG8743. Proteins homologous to a protein encoded by the coding sequence of
  • Drosophila gene CG8743 (SEQ ID NO: 2) include, but are not limited to, the following: Homo sapiens mucolipin-3 protein (GenBank accession No. AAL84622 (SEQ ID NO: 4); having 41 % identity and 61 % homology over 83% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO:2)); a Mus musculus mucolipin-3 (GenBank accession No.
  • AAL84623 (SEQ ID NO: 6); having 40% identity and 60% homology over 86% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO: 2)); a Mus musculus unnamed, putative mucolipidin (Genbank accession No. BAB29372 (SEQ ID NO: 8); having 39% identity and 60%> homology over 78% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO: 2)); a C. elegans hypothetical protein R13A5.1 a (GenBank accession No.
  • AAL38964 (SEQ ID NO: 10); having 37% identity and 59% homology over 84% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO: 2)); a C. elegans hypothetical protein R13A5.1 a.p (GenBank accession No. NP 498664 (SEQ ID NO: 12); having 37% identity and 59% homology over 84% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO: 2)); a C. elegans hypothetical protein R13A5.1 b.p (GenBank accession No.
  • NP_498665 (SEQ ID NO: 14)); having 37% identity and 59% homology over 84% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO: 2)); a Homo sapiens mucolipidin protein (GenBank accession No. CAC0821 5 (SEQ ID NO: 16)); having 38% identity and 58% homology over 85% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO:2)) ; a C. elegans CUP-5L protein (GenBank accession No.
  • AAL89754 (SEQ ID NO: 18); having 37% identity and 59% homology over 84% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO: 2)); a C. elegans CUP-5 protein (GenBank accession No. AAK1 9624 (SEQ ID NO: 20); having 37% identity and 59% homology over 84% of the protein encoded by the coding sequence of Drosophila gene CG8743 SEQ ID NO: 2)); a C. elegans hypothetical protein R1 3A5.1 b (GenBank accession No.
  • AAL38965 (SEQ ID NO: 22); having 36% identity and 58% homology over 85% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO: 2)); a Mus musculus unnamed protein similar to mucolipin (GenBank accession No. AAH05651 (SEQ ID NO: 24); having 38% identity and 58% homology over 85% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO: 2)); a Homo sapiens mucolipin 1 protein (GenBank accession No. AAG42242 (SEQ ID NO: 26); having 37% identity and 56% homology over
  • XP_088847 (SEQ ID NO: 32); having 35% identity and 52% homology over 77% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO: 2)); a Mus musculus mucolipin-3 protein (Genbank accession No. NP_598921 (SEQ ID NO: 34); having 38% identity and 58% homology over
  • AAM1 5597 (SEQ ID NO: 38); having 35% identity and 56% homology over 84% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO: 2)); a Mus musculus mucolipin-2 protein (Genbank accession No. AAM28596 (SEQ ID NO: 40); having 37% identity and 58% homology over 78% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO: 2)); a Mus musculus RIKEN cDNA 3300002C04 protein (Genbank accession No.
  • NP 080932 (SEQ ID NO: 42); having 37% identity and 58% homology over 78% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO: 2)); a Homo sapiens mucolipin 1 protein (GenBank accession No. NP 065394 (SEQ ID NO 44; having 36% identity and 56% homology over 85% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO: 2)); a Mus musculus mucolipin 1 protein (GenBank accession No.
  • NP_444407 SEQ ID NO: 46; having 36% identity and 56% homology over 85% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO:2)); a Homo sapiens mucolipin-3 protein (GenBank accession No. NP 060768 (SEQ ID NO: 48); having 36% identity and 53% homology over 83% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO 2)); a Homo sapiens mucolipidin protein (GenBank accession No.
  • CAC07813 (SEQ ID NO: 50); having 40% identity and 59% homology over 54% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO: 2)); a C. elegans CUP-5 protein (GenBank accession No. AAM1 5596 (SEQ ID NO: 52); having 35% identity and 56% homology over 84% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO: 2)); a Mus musculus mucolipin-1 protein (GenBank accession No.
  • AAL58667 (SEQ ID NO: 54); having 36% identity and 56% homology over 85% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO: 2)); a Homo sapiens mucolipin 1 protein (GenBank accession No. AAH05149 (SEQ ID NO: 56); having 36% identity and 56% homology over 85% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO: 2)); a Homo sapiens mucolipin protein (GenBank accession No.
  • AAG00798 (SEQ ID NO: 58); having 36% identity and 56% homology over 85% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO: 2)); a Homo sapiens mucolipin protein (GenBank accession No. AAG00797 (SEQ ID NO: 60); having 36% identity and 56% homology over 85% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO: 2)); a Mus musculus putative homolog to mucolipidin (GenBank accession No.
  • BAB31730 (SEQ ID NO: 62); having 37% identity and 58% homology over 78% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO: 2)); and a Homo sapiens mucolipidosis type IV protein (GenBank accession No. AAG 10422 (SEQ ID NO: 64); having 36% identity and 56% homology over 85% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO: 2)).
  • other proteins that may be used in the methods provided herein include, but are not limited to, proteins involved in intracellular calcium modulation that are substantially homologous to the above-listed proteins.
  • the agent administered to the subject is one that modulates the level and/or activity of a mucolipidin (also referred to as mucolipin) or mucolipidin-like protein.
  • mucolipidin or mucolipidin-like proteins include the following: Homo sapiens mucolipin-3 protein (GenBank accession No. AAL84622 (SEQ ID NO: 4); having 41 % identity and 61 % homology over 83% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO: 2)); a Mus musculus mucolipin-3 (GenBank accession No.
  • AAL84623 (SEQ ID NO: 6); having 40% identity and 60% homology over 86% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO: 2)); a Mus musculus unnamed, putative mucolipidin (GenBank accession No. BAB29372 (SEQ ID NO: 8); having 39% identity and 60% homology over 78% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO: 2)); a Homo sapiens mucolipidin protein (GenBank accession No.
  • CAC0821 5 (SEQ ID NO: 16); having 38% identity and 58% homology over 85% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO: 2)); a Mus musculus unnamed protein similar to mucolipin 1 protein (GenBank accession No. AAH05651 (SEQ ID NO: 24); having 38% identity and 58% homology over 85% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO: 2)); a Homo sapiens mucolipin 1 protein (GenBank accession No.
  • AAG42242 (SEQ ID NO: 26); having 37% identity and 56% homology over 87% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO: 2)); a Homo sapiens mucolipidin protein (GenBank accession No. CAC07813 (SEQ ID NO: 30); having 43% identity and 63% homology over 54% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO: 2)); a Mus musculus mucolipin-3 protein (GenBank accession No.
  • NP_598921 (SEQ ID NO: 34); having 38% identity and 58% homology over 86% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO: 2)); a Mus musculus mucolipin-2 protein (GenBank accession No. AAM28596 (SEQ ID NO: 40); having 37% identity and 58% homology over 78% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO: 2)); a Homo sapiens mucolipin 1 protein (GenBank accession No.
  • NP 065394 (SEQ ID NO: 44); having 36% identity and 56% homology over 85% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO: 2)); a Mus musculus mucolipin 1 protein (GenBank accession No. NP_444407 (SEQ ID NO: 46); having 36% identity and 56% homology over 85% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO: 2)); a Homo sapiens mucolipin-3 protein (GenBank accession No.
  • NP 060768 SEQ ID NO: 48; having 36% identity and 53% homology over 83% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO: 2)); a Homo sapiens mucolipidin protein (GenBank accession No. CAC07813 (SEQ ID NO: 50); having 40% identity and 59% homology over 54% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO: 2)); a Mus musculus mucolipin-1 protein (GenBank accession No.
  • AAL58667 (SEQ ID NO: 54); having 36% identity and 56% homology over 85% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO:2)); a Homo sapiens mucolipin 1 protein (GenBank accession No. AAH05149 (SEQ ID NO: 56); having 36% identity and 56% homology over 85% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO: 2)); a Homo sapiens mucolipin protein (GenBank accession No.
  • AAG00798 (SEQ ID NO: 58); having 36% identity and 56% homology over 85% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO: 2); a Homo sapiens mucolipin protein (GenBank accession No. AAG00797 (SEQ ID NO: 60); having 36% identity and 56% homology over 85% of the protein encoded by the coding sequence of
  • Drosophila gene CG8743 (SEQ ID NO: 2)); a Mus musculus putative homolog to mucolipidin (GenBank accession No. BAB31730 (SEQ ID NO: 62); having 37% identity and 58% homology over 78% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO: 2)); and a Homo sapiens mucolipidosis type IV protein (GenBank accession No. AAG 10422 (SEQ ID NO: 64); having 36% identity and 56% homology over 85% of the protein encoded by the coding sequence of Drosophila gene CG8743 (SEQ ID NO: 2).
  • the diseases and disorders provided herein include, but are not limited to, proteins involved in intracellular calcium modulation that are substantially homologous to the above-listed proteins and mutant or polymorphic forms thereof.
  • the disease or disorder can be, for example, a neurodegenerative disease or disorder, e.g., Alzheimer's disease (AD), Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis (ALS), and other brain disorders caused by trauma or other insults including aging, an inflammatory disease (e.g.
  • Neurodegenerative diseases and disorders include but are not limited to Alzheimer's disease (AD), Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis (ALS), and other brain disorders caused by trauma or other insults including aging.
  • AD Alzheimer's disease
  • Parkinson's disease Huntington's disease
  • ALS amyotrophic lateral sclerosis
  • other brain disorders caused by trauma or other insults including aging.
  • Mechanisms associated with calcium signaling may be altered in many neurodegenerative diseases and in disorders resulting from brain injury.
  • fibroblasts or T-lymphocytes from patients with AD have consistently displayed an increase in Ca 2+ release from intracellular stores compared to controls (Ito et al. (1 994) Proc. Natl. Acad. Sci. U.S.A. S /:534-538; Gibson et al. (1 996) Biochem. Biophys. ACTA 1 31 6:71 -77; Etchenberrigaray et al. ( 1 998) Neurobiology of Disease, 5:37-45).
  • PS1 or PS2 presenilin genes associated with familial AD (FAD) have been shown to increase lnsP3-mediated Ca 2+ release from internal stores (Guo et al. (1 996) Neuro Report, 3:379-383; Leissring et al. (1 999) J. Neurochemistry, 72:1061 -1068; Leissring et al. (1 999) J. Biol. Chem. 274(46):32535-32538; Leissring et al. (2000) J. Biol. Chem. 149 (4) :793-797; Leissring et al. (2000) Proc. Natl. Acad. Sci. U.S.A.
  • Methods provided herein may also be used in connection with treatment of inflammatory diseases.
  • diseases include but are not limited to asthma, chronic obstructive pulmonary disease, rheumatoid arthritis, inflammatory bowel disease, glomerulonephritis, neuroinflammatory diseases such as multiple sclerosis, and disorders of the immune system.
  • Methods provided herein may also be used in connection with treatment of malignancies, including, but not limited to, malignancies of lymphoreticular origin, bladder cancer, breast cancer, colon cancer, endometrial cancer, head and neck cancer, lung cancer, melanoma, ovarian cancer, prostate cancer and rectal cancer.
  • Store-operated calcium entry may play an important role in cell proliferation in cancer cells (Weiss et al. (2001 ) International Journal of Cancer 92 t6/:877-882).
  • a major function of prostaglandins is to protect the gastric mucosa. Included in this function is the modulation of store-operated calcium influx in human gastric cells which plays a critical role in cell proliferation.
  • NSAIDs nonsteroidal anti-inflammatory drugs
  • the NSAIDs that relieve inflammation most effectively also produce the greatest gastrointestinal damage (Canadian Family Physician, January 1998, p. 101 ).
  • the ability to independently modulate store-operated calcium channels in specific cell types may help to alleviate such side effect of anti-inflammatory therapy.
  • liver diseases and disorders include but are not limited to alcoholic liver disease, liver injury, for example, due to transplantation, hepatitis, cancer, and cirrhosis.
  • Methods provided herein may also be used in connection with treatment of kidney diseases and disorders.
  • Mesangial cell hyperplasia is often a key feature of such diseases and disorders.
  • diseases and disorders may be caused by immunological or other mechanisms of injury, including IgAN, membranoproliferative glomerulonephritis or lupus nephritis. Imbalances in the control of mesangial cell replication also appear to play a key role in the pathogenesis of progressive renal failure.
  • mesangial hyperplasia due to elevated proliferation rate or reduced cell loss of mesangial cells.
  • mesangial cell proliferation is induced without cell loss, for example due to mitogenic stimulation, mesangioproliferative glomerulonephritis can result.
  • regulators of mesangial cell growth may act by regulating store-operated calcium channels (Ma et al. (2001 ) J. Am. Soc. of Nephrology, 12:(1 ) 47-53).
  • Modulators of store-operated calcium influx may aid in the treatment of glomerular diseases by inhibiting mesangial cell proliferation.
  • the epithelial calcium channel CaT2 has also been implicated in hypercalciuria and resultant renal stone formation (Peng et al. (2000) J. Biol. Chem., 275(36/: 28186- 281 94). ,
  • Agents for use in the methods of treating a disease or disorder can be any substance or combination of substances that modulates the level and/or activity of a protein involved in modulating intracellular calcium as provided herein.
  • agents include, but are not limited to, small organic molecules, amino acids, peptides, polypeptides, nucleotides, nucleic acids, polynucleotides, carbohydrates, lipids, lipoproteins and glycoproteins.
  • an agent for treatment of a disease or disorder is a composition, such as a compound or combination of compounds, that when administered to a subject having a disease or disorder effectively reduces, ameliorates or eliminates a symptom or manifestation of the disease or disorder or that cures the disease or disorder.
  • the agent can have such an effect alone or in combination with other agents, or may function to enhance a therapeutic effect of another agent.
  • agents include nucleic acids, proteins, chemical compounds, carbohydrates or lipids. 3.
  • Agents for use in the methods of treating a disease or disorder as provided herein may be delivered to a subject using any methods known in the art or described herein.
  • delivery of an agent involves the administration of an effective amount of agent or a pharmaceutically acceptable salt or derivatives thereof.
  • the agent may be administered with a pharmaceutically acceptable, non-toxic, excipient, including solid, semi-solid, liquid or aerosol dosage forms.
  • Administration of the agent can be via a variety of modes and formulations for administering compounds to subjects.
  • the agent may be administered orally, nasally, intrabronchially, rectally, parenterally, intravascularly, transdermally (including electrotransport), or topically, in the form of a solid, semi-solid, lyophilized powder, or liquid dosage forms, such as, for example, tablets, suppositories, pills, capsules, powders, solutions, suspensions, emulsions, creams, lotions, aerosols, ointments, injectables and gels, preferably in unit dosage forms suitable for simple administration of precise dosages.
  • compositions typically will include a pharmaceutical carrier or excipient and an active compound (i.e., the agent or pharmaceutically acceptable salt or derivative thereof), and, in addition, may include other medicinal agents, pharmaceutical agent carriers, adjuvants and other such substances.
  • a pharmaceutically acceptable, non-toxic composition may be formed by the incorporation of excipients, such as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, talcum, cellulose, glucose, gelatin, sucrose and magnesium carbonate.
  • excipients such as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, talcum, cellulose, glucose, gelatin, sucrose and magnesium carbonate.
  • Such compositions may take several forms, such as solutions, suspensions, tablets, pills, capsules, powders and sustained release formulations.
  • the composition may contain, along with the active ingredient, a diluent, such as lactose, sucrose, dicalcium phosphate, a disintegrant, such as starch or derivatives thereof, a lubricant, such as magnesium stearate, and a binder, such as starch, gum acacia, polyvinylpyrrolidone, gelatin, cellulose and derivatives thereof.
  • a diluent such as lactose, sucrose, dicalcium phosphate
  • a disintegrant such as starch or derivatives thereof
  • a lubricant such as magnesium stearate
  • a binder such as starch, gum acacia, polyvinylpyrrolidone, gelatin, cellulose and derivatives thereof.
  • Liquid formulations may, for example, be prepared by dissolving, dispersing an active compound (for example, about 0.1 % to about 95%, or 0.1 % to about 50%, or about 0.5% to about 20%) and optional pharmaceutical adjuvants in a carrier, such as water, saline, aqueous dextrose, glycerol, and ethanol, to thereby form a solution or suspension.
  • a carrier such as water, saline, aqueous dextrose, glycerol, and ethanol
  • the agent may be mixed with a carrier, such as, for example, an oily ester such as ethyl oleate and isopropyl myristate.
  • a carrier such as, for example, an oily ester such as ethyl oleate and isopropyl myristate.
  • Sterile liquid pharmaceutical compositions, solutions or suspensions can be utilized by, for example, intraperitoneal injection, subcutaneous injection, intramuscular injection or intravenously.
  • Transdermal administration of the agent may be conducted through the use of a patch containing the agent and a carrier that is inert to the agent, is non-toxic to the skin and allows delivery of the agent for systemic absorption into the blood stream via the skin.
  • Carriers for transdermal absorption may include pastes, e.g., absorptive powders dispersed in petroleum or hydrophilic petroleum containing the agent with or without a carrier or a matrix containing the agent; creams and ointments, e.g., viscous liquid or semi-solid emulsions, gels and occlusive devices.
  • an agent is administered to achieve an amount effective for amelioration of symptoms of the disease or disorder.
  • the dose required to obtain an effective amount may vary depending on the agent, formulation, disease or disorder, and individual to whom the agent is administered.
  • Determination of effective amounts may also involve in vitro assays in which varying doses of agent are administered to cells in culture and the concentration of agent effective for ameliorating some or all symptoms is determined in order to calculate the concentration required in vivo. Effective amounts may also be based in in vivo animal studies.
  • Drosophila S2 cells is associated with a reduction in basal or resting cytosolic calcium levels and a reduction in store-operated calcium entry into the cells.
  • the protein encoded by CG8743 and proteins homologous with CG8743, are identified herein as being involved in modulating intracellular calcium.
  • proteins are identified herein as being involved in maintenance of resting cytosolic calcium levels and/or store-operated calcium entry.
  • mucolipidin or mucolipin
  • mucolipidin-like proteins are primarily located in the plasma membrane and intracellular compartments, such as late endosomes and/or lysosomes.
  • MCOLN1 mammalian mucolipidin 1
  • MCOLN1 ML4
  • Mucolipidosis type IV a lysosomal storage disorder which results in progressive neurological degeneration characterized by mental retardation, corneal opacities, and delayed motor milestones (see, e.g. , Bassi et al. (2000) Am. J. Hum. Genet. 67: 1 1 10-1 1 20).
  • ML4 RNA expression has been found in most all tissues including epithelial cells, pancreatic cells, hepatocytes, chondrocytes, renal cells, neurons, fibroblasts, and amniotic cells.
  • epithelial cells pancreatic cells
  • hepatocytes chondrocytes
  • renal cells neurons
  • fibroblasts and amniotic cells.
  • mucolipidin 1 is differentially localized and presumably has different roles in the various tissues in which it is expressed.
  • Mucolipidin orthologs have been localized to predominantly the plasma membrane and in intracellular compartments.
  • Cells of almost every cell type of subjects with MLIV contain cytoplasmic inclusions of sphingolipids, phospholipids and acid mucopolysaccharides referred to as storage bodies, although catabolism of the stored molecules by lysosomal hydrolases appears to be normal.
  • the defect in the cells is associated with a substantial alteration in the kinetics of intracellular transport and thus affects membrane sorting and/or late steps of endocytosis of membrane components (particularly from late endosomes to lysosomes) (Chen et al. (1998) Proc. Natl. Acad. Sci. U.S.A. 35:6373-6378).
  • Cytosolic calcium concentration has been shown to play a significant role in membrane fusion events such as lysosome-plasma membrane fusion (Rodrigues et al. (1 997) J. Cell Biol. 737:93-104)), lysosome-phagosome fusion (Jaconi et al. (1990) J. Cell Biol. 770:1555-1 564), lysosome-lysosome fusion (Bakker et a/. (1997) J. Cell Sci. 1 10:2227-2238), and lysosome-endosome fusion (Pryor et al. (2000) J. Cell Biol. 149: 053-1062).
  • the fusion process may require a release of calcium from intra-organellular stores subsequent to docking of the organelles (Pryor et al. (2000) J. Cell Biol. 743:1053-1062). Because calcium is significantly involved in critical events of the endocytic process, and disruption of the endocytic pathway can result in membrane/protein trafficking and lysosomal storage disease and disorders, disruptions in intracellular calcium may play a role in cell trafficking and lysosomal storage diseases, including MLIV.
  • proteins such as mucolipidin
  • cytosolic calcium and store-operated calcium entry provides the basis for methods of treating diseases and disorders of membrane/protein trafficking and endocytic and lysosomal storage diseases provided herein.
  • the methods include a step of contacting a cell, in particular a cell containing a defect that predisposes the cell to or causes within the cell an accumulation of membrane components, including, for example, lipids (e.g., sphingolipids, phospholipids and acid mucopolysaccharides), with an agent that modulates intracellular calcium.
  • the agent can be any agent known in the art to modulate intracellular calcium or one identified using methods described herein. Such agents include, but are not limited to.
  • the agent is one that modulates cytoplasmic calcium levels or movements and/or modulates store-operated calcium entry.
  • the agent is one that reduces cytoplasmic calcium levels and/or increases store-operated calcium entry.
  • Methods for treating membrane/protein trafficking, endocytic and lysosomal storage diseases and disorders include a step of administering to a subject having such a disease or disorder an agent that modulates intracellular calcium.
  • the agent can be any agent known in the art to modulate intracellular calcium or one identified using methods described herein.
  • the agent is one that modulates cytoplasmic calcium levels or movements and/or modulates store-operated calcium entry.
  • the agent is one that reduces cytoplasmic calcium levels and/or increases store-operated calcium entry.
  • EXAMPLE 1 Gene Selection Candidate intracellular calcium-modulating proteins, and genes encoding such proteins, were selected by searching the annotated Drosophila genome database (GADFLY) (http://hedgehog.lbl.gov:8001 /cgi-bin/annot/query/). Search criteria included annotated molecular function, protein domains containing an ion channel, and primary structures associated with ion transporters.
  • GDFLY Drosophila genome database
  • the Drosophila gene accession number was entered as a query in the ENTREZ protein search command line and, in the results page, the BLink (BLAST Link) feature was used to identify protein across taxonomy that had varying degrees of homology and identity to the Drosophila gene product.
  • the BLink feature provides a display of graphical alignments, editable taxonomic trees, conserved protein classes, protein domains, and 3D structures. A Cut-off value of 1 51 was used to limit the number of alignments by BLAST score to those proteins having at least 35% homology over at least 54% of the protein.
  • RNA Interference RNA Interference
  • RNAi Protocol RNA interference
  • DES Drosophila expression system
  • FBS FBS
  • DES complete media penicillin/streptomycin
  • Double-stranded RNA (dsRNA) production DNA templates (approximately 500-700 bp in length) corresponding to the target genes were purified from bacterial stocks (ESTSD02261 ResGen, Invitrogen Corp., for CG8743) using the Qiagen miniprep system following manufacturers protocols.
  • the target DNA was amplified using the EXPAND Long Template PCR system (Roche) as per manufacturer's protocols with the following primers.
  • the sense strand primer used for PCR amplification contained a 5' T7 RNA polymerase binding site (GAATTAATACGACTCACTATAGGGAGA) followed by sequence specific for the CG8743 gene.
  • Sense-Strand Primer (8743-T7-1 1 5) (44-mer): GAA TTA ATA CGA CTC ACT ATA GGG AGA TTG CTT GTT GCG CCT GT (SEQ ID NO: 65).
  • the antisense-strand primer used for PCR amplification contained a 5' T3 RNA polymerase binding site (AATTAACCCTCACTAAAGGGAGA) followed by sequence specific for the CG8743 gene.
  • Antisense-Strand Primer (8743-T3-647) (40-mer):
  • Double-stranded RNA was generated from the amplification products using the T7 Megascript and T3 Megascript Kits (Ambion, Austin, TX). Duplex RNA was achieved by heating equivalent amounts of sense and antisense stands to 65°C for 30 minutes and then slow cooling to room temperature. The concentration of dsRNA was determined by spectrophotometry (optical density at 260 nm). Double-stranded RNA was stored at -20°C. Conditions for RNAi in Drosophila cell culture Drosophila cell culture cells were diluted to a final concentration of 1 X 10 6 cells/ml in DES complete medium. Six milliliters of cells were plated in a 75 cm 2 T-flask.
  • the cells were allowed to attach to the flask, and the DES complete media was replaced with 6 ml DES serum-free media.
  • dsRNA was added directly to the media to an approximate final concentration of 37 nM and mixed by agitation.
  • the cells were incubated for 30 min at room temperature followed by addition of 1 2 ml of DES complete media.
  • the cells were treated with the dsRNA on day 0 of the time course.
  • the cells were incubated and analyzed over a 4-day period to determine turnover of the target gene. Analysis of gene silencing by western blot and RT-PCR
  • Cell extract preparation Cells were harvested by trituration and pelleted by centrifugation at 1000 x g. Cells ( 1 x 10 6 ) were lysed in 100 ⁇ l lysis buffer (50 mM Tris, pH 8.0; 1 50 mM NaCI; 1 .0% Triton; 5 mM EDTA; 0.2% Tween 20). Lysed cells were centrifuged at 1 3,000 x g for 30 min at 4°C to pellet cell debris. The supernatant containing the whole cell extract was stored at -20°C. Analysis and results
  • RNAi-mediated silencing of CG8743 For analysis of RNAi-mediated silencing of CG8743, RT-PCR alone was used to monitor mRNA levels.
  • the 4-day time course experiment was designed to monitor calcium homeostasis (see below) and CG8743 message levels relative to control cells each day. Reduction in CG8743 mRNA levels was observed starting on day one. CG8743 mRNA levels were reduced to less than 10% of the loading control (Drosophila presenilin GenBank ACC NO. U77934) levels by day 2. Message levels remained at 10% of control on days 3 and 4 of the time course analysis.
  • Control cells and cells subjected to RNAi for the silencing of CG8743 were analyzed for possible effects of gene silencing on cytosolic calcium. Specifically, basal cytosolic calcium levels and store-operated calcium entry into the cells were evaluated using a fluorescence-based assay. Fluorescence-based assay protocol
  • S2 cells were seeded in a 96-well plate at a density of 100,000 cells/well (100 l of 10 6 cells/ml stock). The following day, the media was removed and cells were washed with 2 mM Ca 2 + buffer (1 20 mM NaCI, 5 mM KCI, 4 mM
  • Probenecid inhibits the multidrug resistance transporter, which otherwise would transport loaded dye out of the cell.
  • Fluorescence of the dye-loaded cells was measured using an excitation wavelength of 485 nm and an emission wavelength of 510 nm on a FluoroSkan fluorimeter (Lab Systems). Both an Fmax and total fluorescence were measured for each well.
  • the Fmax is the fluorescence of lysed cells that is measured 10 minutes after adding 10 l of 1 % Triton X-100 to each well.
  • the Fmax provides a measure of the relative amount of dye loaded in each well of the 96-well plate, and is proportional to the total number of cells in each well.
  • Total fluorescence is the absolute fluorescence of a well. Evaluation of basal cytosolic calcium
  • Initial basal, resting calcium levels were determined by measuring the fluorescence of the wells prior to adding any type of agent (e.g., store-depletion agent) or manipulating the medium (e.g., altering the calcium concentration of the medium) in order to evaluate store-operated calcium entry as described below.
  • agent e.g., store-depletion agent
  • manipulating the medium e.g., altering the calcium concentration of the medium
  • the total fluorescence of a well was divided by the Fmax for that well.
  • the total fluorescence/Fmax values of different wells containing control cells and cells subjected to RNAi to silence CG8743 gene expression were compared.
  • Store-operated calcium channels have specific pharmacological characteristics that are distinct from other ion channels.
  • Store-operated calcium channels are activated in response to depletion of intracellular calcium stores, which is one feature that distinguishes them from other ion channels such as voltage-gated channels.
  • Store-operated calcium entry can, therefore, be monitored by using store depletion to activate channel activity.
  • Passive or active store depletion may be used to activate store-operated channel activity.
  • Passive intracellular store depletion can be achieved using a buffering agent such as EGTA or chelator such as TPEN.
  • Active intracellular Ca 2+ store depletion can be achieved using inositol-1 ,4,5-triphosphate (lnsP 3 ) to release Ca 2+ from the stores directly.
  • Thapsigargin or thapsigargin-like compounds and compounds such as ionomycin, BHQ, and cyclopiazonic acid may also be used to actively deplete Ca 2+ from intracellular stores.
  • Store-operated calcium entry into S2 cells was evaluated in terms of cytosolic calcium levels determined by measuring the fluorescence of the wells after addition of a store-depletion agent.
  • Cells were incubated with either 10 ⁇ l of 0.1 % DMSO (control) or 10 ⁇ l of 10 M thapsigargin for 5 minutes at room temperature.
  • Thapsigargin acts to inhibit the ER Ca 2+ pump and discharges intracellular Ca 2+ stores.
  • thapsigargin elicits a transient elevation in cytosolic free Ca 2+ concentration ((Ca 2"1" ),), which is believed to result from release of stored Ca 2+ , followed by removal or buffering of cytosolic Ca 2+ back to prestimulation levels.
  • the FLUO-4 assay protocol was conducted in a time-dependent manner over the course of the four days after contacting cells with dsRNA in performing the RNAi protocol.
  • the decrease in store-operated calcium entry was observed on day 2 and reached a maximum on day 3 which lasted into day 4.
  • the increase in basal, resting cytosolic calcium levels was seen on day 2 and continued to increase over each successive day of the time course reaching 1 50% of control levels at day 4, indicating that the two effects (i.e., increase basal cytosolic calcium levels and decreased store-operated calcium entry) are independent.
  • Cells that contain one or more proteins involved in intracellular calcium modulation are used to screen for agents that modulate intracellular calcium.
  • Transfection Untransfected cells as well as recombinant or transfected cells can be used with any of the methods and process described herein.
  • any methods known to those of skill in the art for the insertion of DNA fragments into a vector may be used to construct expression vectors containing nucleic acid encoding an intracellular calcium-modulating protein, and appropriate transcriptional/translational control signals and/or other protein-encoding sequences.
  • DNA inserts may include in vitro recombinant DNA and synthetic techniques and in vivo recombinants (genetic recombination).
  • Nucleic acid encoding an intracellular calcium-modulating protein may be introduced into host cells using a variety of procedures which are known in the art and described herein.
  • Transfection methods include, but are not limited to, lipid-mediated transfer and calcium phosphate precipitation; however, the particular procedure used will depend in part on the host cell. Examples of particular transfection procedures include direct uptake using calcium phosphate (CaP0 4 ; see, e.g., Wigler et al. (1979) Proc. Natl. Acad. Sci. U.S.A. 76:1 373- 1376), polyethylene glycol (PEG)-mediated DNA uptake, electroporation, lipofection (see, e.g., Strauss (1996) Meth. Mol. Biol.
  • CaP0 4 calcium phosphate
  • PEG polyethylene glycol
  • microcell fusion see, e.g., Lambert (1 991 ) Proc. Natl. Acad. Sci. U.S.A. 33:5907-591 1 ; U.S. Patent No. 5,396,767, Sawford et al. (1987) Somatic Cell Mol. Genet. /3:279-284; Dhar et al. (1984) Somatic Cell Mol. Genet. 70:547-559; and McNeill-Killary et al. (1995) Meth. Enzymol. 254:133-152), lipid-mediated carrier systems (see, e.g., Teifel et al. (1 995) Biotechniques 19:79-80; Albrecht et al.
  • RNAi or gene knockout methods may be used to suppress this activity.
  • Other methods of suppression include but are not limited to treatment with sense and anti-sense nucleotides and monoclonal antibodies.
  • Such techniques may be accomplished in accordance with any of the known procedures for treating cells to control the production or expression of a selected protein. Such techniques may be implemented prior to or after transfection of cells or may be incorporated into the transfection procedure.
  • Test agent screening protocol Agents to be screened
  • SH-SY5Y Cell Assay In an exemplary protocol for conducting an agent screening method, human neuroblastoma cells of cell line SH-SY5Y were evaluated for store- operated calcium entry using fluorescence measurement assays as follows. SH- SY5Y cells were plated in 384-well plates were loaded for 45 min with FLUO-4- AM in a Hanks-buffered salt solution. Cells were washed and placed in a nominally Ca 2 + -free Hanks solution. One minute later, 1 ⁇ M thapsigargin (TG) was added to inhibit the ER Ca 2+ pump and discharge intracellular Ca 2+ stores.
  • TG thapsigargin
  • store depletion may be carried out by any of the methods described herein or known in the art. Fifteen minutes after addition of TG, test compound or vehicle was added, followed by another 15 min incubation in Ca 2+ - free buffer. Store-operated calcium entry was then initiated by adding external Ca 2+ to a final concentration of 1 .8 mM and the response monitored using a FLIPR 384 (Molecular Devices flourimetric imaging plate reader for high throughput screening) over a period of 10-15 min.
  • FLIPR 384 Molecular Devices flourimetric imaging plate reader for high throughput screening
  • the kinetic data from the FLIPR 384 was analyzed and then stored in a relational database (ActivityBase; IDBS).
  • ActiveBase a relational database
  • Ten quantitative parameters were calculated that define various aspects of the store-operated calcium entry response. These parameters were as follows:
  • basal fluorescence relative fluorescence units
  • Up slope linear regression of the increase in RFU from 2 to 60 sec after addition of Ca + Up rate constant (Up K): the rate constant derived from first-order association of RFUs from 2 seconds to peak response
  • Peak the peak RFU (single point) achieved after addition of Ca 2+ (this parameter, although calculated, is not returned to the database)
  • Peak/Basal the difference between peak and mean basal RFU
  • Decay slope linear regression of the decrease in RFU from the peak to the end of the measurement period
  • Decay rate constant (Decay K): the rate constant derived from first-order decay of RFUs from the peak to the end of the measurement period.
  • AUC Area under the curve
  • Combinations of these parameters were queried to identify agents that produce at least a 50% change from control. Active agents identified from these queries were retested under identical conditions to confirm their activity.
  • CHO Cell Assay CHO cells transfected with DNA encoding wild-type human APP695 and wild-type human presenilin-1 were treated in a manner similar to the SH-SY5Y cells, except that the additions were performed at different times. Cells loaded with FLUO-4, were incubated in nominally Ca 2+ -free buffer and treated with 1 ⁇ M TG for 5 minutes prior to addition of vehicle or test compound. Following a five minute incubation with compound or vehicle, store-operated calcium entry is initiated by the addition of 1 .8 mM Ca 2+ . Since modifications will be apparent to those of skill in this art, it is intended that this invention be limited only by the scope of the appended claims.

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Abstract

L'invention concerne des procédés pour identifier les agents qui modulent le calcium intracellulaire. Elle concerne également des procédés pour moduler le calcium à l'intérieur des cellules et traiter des maladies par la modulation du calcium intracellulaire, de même que des procédés pour cribler les acides nucléiques à la recherche des protéines candidates de transport d'ions et des procédés pour identifier les acides nucléiques codant les protéines de transport d'ions.
PCT/US2003/026409 2002-08-20 2003-08-20 Procede pour moduler ou identifier les agents qui modulent le calcium intracellulaire WO2004018700A2 (fr)

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MAASCH C. ET AL.: 'Protein kinase c-alpha targeting is regulated by temporal and spatial changes in intracellular free calcium concentration' FASEB J vol. 14, 2000, pages 1653 - 1663 *

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