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WO2002040534A1 - Sequences nucleotidiques et polypeptidiques de kcna7, une proteine mammalienne du canal potassique potentiel-dependant, et utilisations de ces dernieres - Google Patents

Sequences nucleotidiques et polypeptidiques de kcna7, une proteine mammalienne du canal potassique potentiel-dependant, et utilisations de ces dernieres Download PDF

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WO2002040534A1
WO2002040534A1 PCT/SE2001/002552 SE0102552W WO0240534A1 WO 2002040534 A1 WO2002040534 A1 WO 2002040534A1 SE 0102552 W SE0102552 W SE 0102552W WO 0240534 A1 WO0240534 A1 WO 0240534A1
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kcna7
polypeptide
nucleic acid
candidate agent
mammalian
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PCT/SE2001/002552
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Vladimir I. Kashuba
Claes Wahlestedt
Eugene R. Zabarovsky
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Karolinska Innovations Ab
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Priority to AU2002214522A priority Critical patent/AU2002214522A1/en
Publication of WO2002040534A1 publication Critical patent/WO2002040534A1/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
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/502—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects
    • G01N33/5023—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects on expression patterns
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • 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
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • 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
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/502—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects
    • 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)
    • 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
    • G01N2500/10—Screening for compounds of potential therapeutic value involving cells
    • 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
    • G01N2500/20—Screening for compounds of potential therapeutic value cell-free systems

Definitions

  • the present invention relates to novel nucleic acid molecules coding for a mammalian voltage-gated potassium channel protein.
  • the invention further relates to methods for identifying agents capable of modulating voltage-gated potassium ion channel activity.
  • Kv channels Noltage-gated potassium ion channels
  • the largest sub-family of the ion channel superfamily play important roles in a wide variety of cells (for reviews, see Choe, S. et al. (1999) Trends Biochem. Sci. 24: 345-349; Lehmann-Horn, F. & Jurkat- Rott, K. (1999) Physiological Reviews 79: 1317-1372).
  • Membrane depolarization activates voltage-gated potassium channels that, once opened, conduct potassium ions along the concentration gradient against the electric field. This outward current leads to repolarization of the membrane.
  • Kv channels in mammalian cells are encoded by an extended family of at least nineteen genes.
  • the largest subfamily, Kvl is related to the fly Shaker gene and contains at least seven members, Kvl .1— Kvl .7.
  • the mammalian voltage-gated S/z £er-related potassium-channel gene Kvl.7 (Kalman K. et al. (1998) J. Biol. Chem. 273: 5851-5857; see also US patent No. 5,559,009) has been mapped to mouse chromosome 7 and human chromosome 19ql3.3, a region that has been suggested to contain a diabetic susceptibility locus.
  • Kv ion channels are in part responsible for the maintenance of cellular electrical activity.
  • An imbalance in electrical activity is thought to be an underlying cause, at least — -_. —
  • ion channels may be useful targets for discovering ligands or drugs to treat many diverse disorders and defects, including schizophrenia, depression, anxiety, attention deficit hyperactivity disorder, migraine, stroke, ischemia, and neurodegenerative disease such as Alzheimer's disease, Parkinson's disease, glaucoma and macular degeneration.
  • compounds which modulate ion channels can be used for the treatment of cardiovascular diseases including ischemia, congestive heart failure, arrhythmia, high blood pressure and restenosis.
  • the KCNA7 gene (A) Genomic structure of KCNA7, including a putative promoter and the exon positions. (B) Computational detection of potential skeletal muscle regulatory regions (boxes marked "Unknown TF", cf. positions 105 to 114 and 201 to 211 in SEQ ID NO: 13) in human KCNA7 gene and putative mouse sequence. Potential Spl and Mef2 binding sites are also shown (cf. positions 151 to 159 and 183 to 192 in SEQ ID NO: 13).
  • Fig. 3 Hybridization of the KCNA7 to a Human Multiple Tissue Northern blot.
  • the present invention is directed to a novel, putative member of the mammalian voltage-gated potassium channel protein family, the potassium channel KCNA7.
  • the potassium channel KCNA7 We report the cloning of the human ortholog to the murine Kvl .7 potassium ion channel, the tissue distribution of its expression, and the analysis of the genomic sequence encoding the gene.
  • the maximal open reading frame in the human gene encodes a protein of 456 amino acids (SEQ ID NO: 2).
  • the predicted product exhibits 91% amino acid identity to the murine voltage-gated potassium channel Kvl.7 (Kcna7; SEQ ID NO: 6), which plays an important role in the repolarization of cell membranes.
  • Kcna7 murine voltage-gated potassium channel
  • the human protein has been classified as the ortholog of the mouse gene and designated KCNA7.
  • a structural prediction identified a pore region characteristic of potassium channels and a transmembrane segment of the cyclic nucleotide gated channel.
  • Northern expression analysis revealed the gene is expressed preferentially in skeletal muscle and heart. A single RNA isoform was observed, with a size of approximately 4 kb.
  • the gene was mapped to chromosomal band 19ql3.3.
  • a genomic sequence was identified in the database from this region, and the KCNA7 gene structure determined. Computational analysis of the genomic sequence reveals the location of a putative promoter and a likely muscle-specific regulatory region.
  • this invention provides an isolated nucleic acid molecule selected from:
  • nucleic acid molecules comprising a nucleotide sequence as shown in SEQ ID NO: 1 or 11;
  • nucleic acid molecules comprising a nucleotide sequence capable of hybridizing, under stringent hybridization conditions, to a nucleotide sequence complementary the polypeptide coding region of a nucleic acid molecule as defined in (a) and which codes for a biologically active KCNA7 polypeptide or a functionally equivalent modified form thereof;
  • nucleic acid molecules comprising a nucleic acid sequence which is degenerate as a result of tl e genetic code to a nucleotide sequence as defined in (a) or (b) and which codes for a KCNA7 polypeptide or a functionally equivalent modified form thereof.
  • nucleic acid molecules according to the present invention includes cDNA, chemically synthesized DNA, DNA isolated by PCR, genomic DNA, and combinations thereof. RNA transcribed from DNA is also encompassed by the present invention.
  • the said nucleic acid molecule has a nucleotide sequence identical with SEQ ID NOS: 1 or 11 of the Sequence Listing.
  • the nucleic acid molecule according to the invention is not to be limited strictly to the sequence shown as SEQ ID NO: 1 or 11. Rather the invention encompasses nucleic acid molecules carrying modifications like substitutions, small deletions, insertions or inversions, which nevertheless encode proteins having substantially the biochemical activity of the mammalian KCNA7 polypeptide according to the invention.
  • nucleic acid molecules the nucleotide sequence of which is at least 90% homologous, preferably at least 95% homologous, with tl e nucleotide sequence shown as SEQ ID NO: 1 or 11 in the Sequence Listing.
  • nucleic acid molecule which nucleotide sequence is degenerate, because of the genetic code, to the nucleotide sequence shown as SEQ ID NO: 1 or 11.
  • the invention also provides an isolated polypeptide encoded by the nucleic acid according to claim 1.
  • the said polypeptide has an amino acid sequence according to SEQ ID NO: 2 or 12 of the Sequence Listing.
  • the polypeptide according to the invention is not to be limited strictly to a polypeptide with an amino acid sequence identical with SEQ ID NO: 2 or 12 in the Sequence Listing. Rather the invention encompasses polypeptides carrying modifications like substitutions, small deletions, insertions or inversions, which polypeptides nevertheless have substantially the biological activities of mammalian KCNA7.
  • the invention provides a vector harboring the nucleic acid molecule as defined above.
  • vector refers to any carrier of exogenous DNA that is useful for transferring the DNA to a host cell for replication and/or appropriate expression of the exogenous DNA by the host cell.
  • the said vector can e.g. be a replicable expression vector, which carries and is capable of mediating the expression of a DNA molecule according to the invention.
  • replicable means that the vector is able to replicate in a given type of host cell into which is has been introduced.
  • examples of vectors are viruses such as bacteriophages, cosmids, plasmids and other recombination vectors. Nucleic acid molecules are inserted into vector genomes by methods well known in the art.
  • a cultured host cell harboring a vector according to the invention.
  • a host cell can be a prokaryotic cell, a unicellular eukaryotic cell or a cell derived from a multicellular organism.
  • the host cell can thus e.g. be a bacterial cell such as an E. coli cell; a cell from yeast such as Saccharomyces cervisiae or Pichia pastoris, or a mammalian cell.
  • the methods employed to effect introduction of the vector into the host cell are standard methods well known to a person familiar with recombinant DNA methods.
  • a further aspect of the invention is a process for production of a mammalian KCNA7 polypeptide which comprises culturing a host cell as defined above under conditions whereby said polypeptide is produced, and recovering said polypeptide.
  • this invention provides a method for identifying an agent capable of modulating voltage-gated potassium ion channel activity, comprising
  • agent means a biological or chemical compound such as a simple or complex organic molecule, a peptide, a protein or an oligonucleotide.
  • such a method can comprise the steps (i) contacting a candidate agent with a nucleic acid molecule according to the invention, or with the encoded mammalian KCNA7 polypeptide; and (ii) determining whether said candidate agent modulates the expression of the said nucleic acid molecule, or whether the candidate agent modulates the biological activities of the said polypeptide.
  • biological activities is intended to encompass triggering release or uptake of protein or non- protein molecules from the cell, or triggering the opening of the channel and the movement of ions across the cellular membrane, and the electrical signal which accompanies the passage of the ions across the membrane as assessed with the technique of electrophysiology. For example, activity can be determined by measuring ion flux.
  • ion flux includes ion current.
  • Activity can also be measured by measuring changes in membrane potential using electrodes or voltage- sensitive dyes, or by measuring neuronal or cellular activity such as action potential duration or frequency, the threshold for stimulating action potentials, long-term potentiation, or long-term inhibition.
  • Electronal or cellular activity such as action potential duration or frequency
  • the threshold for stimulating action potentials long-term potentiation, or long-term inhibition.
  • Blockers of the mammalian KCNA7 ion channel would be expected to increase insulin release and thereby reduce hyperglycemia associated with non-insulin-dependent diabetes mellitus. Consequently, agents modulating the mammalian KCNA7 gene or KCNA7 protein could be useful for the treatment of diabetes and related medical conditions.
  • modulators of the mammalian KCNA7 ion channel could be useful in the treatment of ion channel related problems such as schizophrenia, depression, anxiety, attention deficit hyperactivity disorder, migraine, stroke, ischemia, glaucoma, macular degeneration, epilepsy, and neurodegenerative disease such as Alzheimer's disease and Parkinson's disease.
  • appropriate host cells can be transformed with a vector having a reporter gene under the control of the KCNA7 gene according to this invention.
  • the expression of the reporter gene can be measured in the presence or absence of an agent with known activity (i.e. a standard agent) or putative activity (i.e. a "test agent” or “candidate agent”).
  • a change in tl e level of expression of the reporter gene in the presence of the candidate agent is compared with that effected by the standard agent. In this way, active agents are identified and their relative potency in this assay determined.
  • reporter gene means a gene encoding a gene product that can be identified using simple, inexpensive methods or reagents and that can be operably linked to the KCNA7 gene or an active fragment thereof.
  • Reporter genes such as, for example, a luciferase, ⁇ -galactosidase, alkaline phosphatase, or green fluorescent protein reporter gene, can be used to determine transcriptional activity in screening assays according to the invention (see, for example, Goeddel (ed.), Methods Enzymol., Vol. 185, San Diego: Academic Press, Inc. (1990); see also Sambrook, supra).
  • the effect of candidate agents on the KCNA7 potassium channel can be monitored by methods known in the art. For instance, the rate of 86 Rb efflux from a 86 Rb loaded cell, expressing the mammalian KCNA7 ion channel, can be monitored (cf. Example 7, below).
  • the candidate agent is a polypeptide
  • its interaction with the KCNA7 polypeptide can be monitored by well known methods for determination of protein- protein interactions. Examples of such methods, applicable for the soluble portion of KCNA7, are the yeast two-hybrid system and FRET (fluorescence resonance energy transfer) (cf. Examples 8 and 9, respectively).
  • FRET fluorescence resonance energy transfer
  • the invention provides a method for the identification of an agent modulating transcription of the human KCNA7 gene, said method comprising the steps (i) contacting a candidate agent with a regulatory region shown as positions 102 to 246, or a part thereof, such as in particular positions 105 to 114 or 201 to 211, in SEQ ID NO: 13; and
  • the novel molecules identified by the screening methods according to the invention are low molecular weight organic molecules, in which case a composition or pharmaceutical composition can be prepared thereof for oral intake, such as in tablets.
  • a composition or pharmaceutical composition comprising the nucleic acid molecules, vectors, polypeptides, antibodies and compounds identified by the screening methods described herein, can be prepared for any route of administration including, but not limited to, oral, intravenous, cutaneous, subcutaneous, nasal, intramuscular or intraperitoneal.
  • the nature of the carrier or other ingredients will depend on the specific route of administration and particular embodiment of the invention to be administered. Examples of techniques and protocols that are useful in this context are, inter alia, found in Remington's Pharmaceutical Sciences, 16 th edition, Osol, A. (ed.), 1980, which is incorporated herein by reference in its entirety.
  • the dosage of these low molecular weight compounds will depend on tl e disease state or condition to be treated and other clinical factors such as weight and condition of the human or animal and the route of administration of the compound.
  • tl e disease state or condition to be treated and other clinical factors such as weight and condition of the human or animal and the route of administration of the compound.
  • For treating human or animals between approximately 0.5 mg/kg of body weight to 500 mg/kg of body weight of the compound can be administered. Therapy is typically administered at lower dosages and is continued until the desired therapeutic outcome is observed.
  • cDNA library from heart (Stratagene, La Jolla, CA, USA) in ⁇ ZAP II was used for the screening and isolation of cDNA clones.
  • Marathon-ReadyTM cDNA from skeletal muscle (Clontech, Palo Alto, CA, USA) was used for 5'- and 3'-RACE PCR.
  • Examples 1 to 5 are actual, while Examples 6 to 10 are prophetic.
  • Notl linking libraries were described previously (Zabarovsky et al. (1994) Genomics 20: 312-316; Zabarovsky et al. (2000) Nucleic Acids Research 28: 1635-1639).
  • the Notl linking clone ⁇ R1-253 (SEQ ID NO: 3; GenBank Accession No. AQ939522, approximately 900 bp) displayed 85% identity over 658 nucleotides with the murine voltage-gated potassium channel Kvl .7 (Kcna7) (SEQ ID NO: 4; GenBank Accession No. AF032099; Kalman, K. et al. (1998) J. Biol. Chem. 273: 5851-5857).
  • a set of overlapping human cDNA sequences was obtained via a combination of cDNA library screening and RACE-PCR according to standard methods.
  • the final full-length human sequence (4372 bp, SEQ ID NO: 1) was obtained by the fusion of the longest 3 '- and 5 '-RACE PCR products with cDNA clone (p5kvl -7) containing the complete ORF.
  • the KCNA7 nucleotide sequence encodes a deduced protein of 456 amino acids (SEQ ID NO: 2).
  • the human KCNA7 gene is split into two exons of length 911 and 3461 bp, respectively (Fig. 1A; positions 1-911 and 912-4372 in SEQ ID NO: 1).
  • the human gene intron is 1153 bp in length, compared to the reported murine intron of length 1.9 kb (Kalman et al., supra).
  • the human KCNA7 protein displays high level of amino acid identity to the mouse Kcna7 protein (SEQ ID NO: 6) (90% in 452 aa overlap, score 809 bits) and less similarity to a variety of human potassium channel genes ( ⁇ 71% in 411-434 aa overlap). In fact, in many extended regions the human and mouse genes are identical (Fig. 2). Based on the observed similarity, we postulate that the human and murine genes are true orthologs. Nucleic acid similarity is less profound, in the best cases reaching 82%-86%, which is consistent with the variability between orthologous human and rodent genes (Makalowski, W. & Boguski, M.S. (1998) Proc. Natl. Acad. Sci. U.S.A. 95(16): 9407-9412).
  • the deduced human KCNA7 protein contains six putative membrane-spanning domains.
  • the region between amino acids 237 and 448 is recognized as a transmembrane part of the cyclic nucleotide gated channel and contains a pore region (amino acids 342-397).
  • the protein contains conserved sites for various post- translational modifications.
  • KCNA7 has a potential tyrosine kinase phosphorylation site (RPSFDAVLY) in its N-terminal region (amino acids 62-70) (Chandy, K.G. & Gutman, G.A.
  • TLR amino acids 304- 306
  • SMR amino acids 308-310
  • the human and murine cDNA sequences indicate different N-terminal sequences in the encoded polypeptides (Fig. 2A).
  • Potassium channels can vary significantly at the 5'- ends, but as these orthologous genes are highly similar, it seems more likely that a frameshift within the murine cDNA sequence could have produced an inaccurate N- terminal sequence.
  • CGGC at positions 383-386 which corresponds to the sequence CGC at positions 523- 525 in the murine cDNA sequence (SEQ ID NO: 5).
  • the murine ESTs for Kcna7 (Accession numbers AI322534.1 (SEQ ID NO: 7) and AJ324179 (SEQ ID NO: 8)) contain the sequence CGGC.
  • a murine genomic sequence (GenBank accession no. AC073711) for the Kcna7 gene, or possibly for a recently created paralog or pseudogene, contains the CGGC sequence.
  • the murine ORF would be altered at the N-terminal such that the first 88 amino acids of the published murine sequence would be replaced by 10 amino acids identical to the human N-terminal sequence. With this correction to the murine sequence, it would show 91% identity with the human sequence (Fig. 2B).
  • Northern hybridization of the cloned human gene was carried out using a filter containing RNA from a variety of muscle tissues (Clontech, Human Muscle #7765-1).
  • Northern expression analysis revealed the highest expression of KCNA7 in skeletal muscle and heart (Fig. 3). A single band of approximately 4 kb was observed. Skeletal muscle is believed to be the principal tissue responding to insulin to modify glucose levels in the body (see e.g. Zierath, J.R. et al. (2000) Diabetologia 43:821-835). Expression in smooth muscles was detected at a lower level consistent with the expression of murine Kcna7.
  • Fluorescence in situ hybridization (FISH) analysis with metaphase chromosomes was performed as described previously (Protopopov et al. (1996) Chromosome Research 4:443-447) .
  • the Notl linking clone ⁇ R1-253, to which the KCNA7 gene corresponds (see Example 1, above) was assigned to chromosomal band 19ql3.3.
  • this map location is consistent with a putative diabetes susceptibility gene that has been suggested to be present at 19ql3.3. This suggestion is especially strong for Finnish families with associated hypertension and difficulties in insulin-stimulated glucose storage (Groop, L.C.
  • vector pCDNA6 For expression of voltage-gated ion channel polypeptides in mammalian cells 293 (transformed human, primary embryonic kidney cells), a plasmid bearing the relevant voltage-gated ion channel coding sequence is prepared, using vector pCDNA6 (Invitrogen).
  • Vector pCDNA6 contains the CMV promoter and a blasticidin resistant gene for selection of stable transfectants. Many other vectors can be used containing, for example, different promoters, epitope tags for detection and/or purification of the protein, and resistance genes.
  • the forward primer for amplification of this voltage-gated ion channel polypeptide encoding cDNA is determined by procedures as well known in the art and preferably contains a 5' extension of nucleotides to introduce the H dIII cloning site and nucleotides matching the voltage-gated ion channel nucleotide sequence.
  • the reverse primer preferably contains a 5' extension of nucleotides to introduce an Xhol restriction site for cloning and nucleotides corresponding to the reverse complement of the voltage-gated ion channel nucleotide sequence.
  • the PCR conditions are 55°C as the annealing temperature.
  • the PCR product is gel purified and cloned into the Hin ⁇ Hl-XhoI sites of the vector.
  • the DNA is purified using Qiagen chromatography columns and transfected into 293 cells using DOTAP transfection media (Boehringer Mannheim, Indianapolis, IN). Transiently transfected cells are tested for expression after 24 hours of transfection, using Western blots probed with anti-His and anti- voltage-gated ion channel peptide antibodies. Permanently transfected cells are selected with Zeocin and propagated. Production of the recombinant protein is detected from both cells and media by western blots probed with anti-His, anti-Myc or anti-voltage-gated ion channel peptide antibodies.
  • a polynucleotide molecule having a nucleotide sequence of SEQ ID NO:l, or complementary nucleotide sequences thereof can be cloned into vector p3-CI.
  • This vector is a pUC 18-derived plasmid that contains the HCMV (human cytomegalo virus) promoter-intron located upstream from the bGH (bovine growth hormone) polyadenylation sequence and a multiple cloning site.
  • the plasmid contains the dhrf (dihydrofolate reductase) gene which provides selection in the presence of the drug methotrexane (MTX) for selection of stable transformants.
  • dhrf dihydrofolate reductase
  • MTX drug methotrexane
  • Many other vectors can be used containing, for example, different promoters, epitope tags for detection and/or purification of the protein, and resistance genes.
  • the forward primer is determined by procedures known in the art and preferably contains a 5' extension which introduces an Xbal restriction site for cloning, followed by nucleotides which correspond to a nucleotide sequence given in SEQ ID NO: 1 , or portion thereof.
  • the reverse primer is also determined by methods well known in the art and preferably contains a 5'- extension of nucleotides which introduces a Sail cloning . site followed by nucleotides which correspond to the reverse complement of a nucleotide sequence given in SEQ ID NO: 1, or portion thereof.
  • the PCR consists of an initial denaturation step of 5 min at 95 °C, 30 cycles of 30 sec denaturation at 95°C, 30 sec annealing at 58°C and 30 sec extension at 72°C, followed by 5 min extension at 72°C.
  • the PCR product is gel purified and ligated into the Xbal and Sail sites of vector p3-CI. This construct is transformed into E. coli cells for amplification and DNA purification.
  • the DNA is purified with Qiagen chromatography columns and transfected into COS 7 cells using Lipofectamine reagent (Gibco/BRL), following the manufacturer's protocols. Forty-eight and 72 hours after transfection, the media and the cells are tested for recombinant protein expression.
  • Voltage-gated ion channel polypeptides expressed in cultured COS cells can be purified by disrupting cells via homogenization and purifying membranes by centrifugation, solubilizing the protein using a suitable detergent, and purifying the protein by, for example, chromatography. Purified voltage-gated ion channel is concentrated to about 0.5 mg/ml in an Amicon concentrator fitted with a YM-10 membrane and stored at -80°C.
  • EXAMPLE 7 Use of Kvl .7 expression construct to identify Kvl .7-specific glucose-dependent insulin secretagogues
  • the KCNA7 expression construct [described above] can be used to generate functional potassium channels with unique properties.
  • This construct can be used for expression of functional KCNA7 channels in mammalian cell lines that do not express endogenous potassium channels (e.g., CV-1, NTH-3T3, or RBL cell lines). These cell lines can then be loaded with s6 Rb (Rb ions permeate through potassium channels nearly as well as potassium ions) in the presence of absence of extrinsic materials, and KCNA7 modifiers identified by their ability to alter 86 Rb-efflux. When natural toxins are identified which block KCNA7 activity, modifiers of KCNA7 activity could also be identified by their ability to block or reverse the binding of labeled toxins to cells expressing this channel. Compounds discovered in either of these manners could then be formulated and administered as therapeutic agents for the treatment of NIDDM.
  • EXAMPLE 8 Interaction Trap/Two-Hybrid System
  • the interaction trap/two-hybrid library screening method can be used. This assay was first described in Fields & Song (1989) Nature 340: 245-246. A protocol is published in Current Protocols in Molecular Biology 1999, John Wiley & Sons, NY, and Ausubel, F.M. et al. 1992, Short Protocols in Molecular Biology, 4 th ed., Greene and Wiley- Interscience, NY. Kits are available from Clontech, Palo Alto, CA (Matchmaker Two- Hybrid System 3).
  • a fusion of the nucleotide sequences encoding an intracellular, soluble portion of the voltage-gated ion channel polypeptide and the yeast transcription factor GAL4 DNA- binding domain is constructed in an appropriate plasmid (i.e., pGBKT7), using standard subcloning techniques.
  • a GAL4 active domain (AD) fusion library is constructed in a second plasmid (i.e., pGADT7) from cDNA of potential voltage-gated ion channel polypeptide-binding proteins.
  • the DNA-BD/voltage-gated ion channel fusion construct is verified by sequencing, and tested for autonomous reporter gene activation and cell toxicity, both of which would prevent a successful two- hybrid analysis.
  • Yeast cells are transformed (ca. 10 5 transformants/mg DNA) with both the voltage-gated ion channel and library fusion plasmids according to standard procedures. In vivo binding of DNA- BD/voltage-gated ion channel with AD/library proteins results in transcription of specific yeast plasmid reporter genes (i.e., lacZ, HIS 3, ADE2, LEU2). Yeast cells are plated on nutrient-deficient media to screen for expression of reporter genes.
  • yeast plasmid reporter genes i.e., lacZ, HIS 3, ADE2, LEU2
  • Colonies are dually assayed for ⁇ -galactosidase activity upon growth in Xgal (5-bromo-4-chloro- 3-indolyl- ⁇ -D-galactoside) supplemented media (filter assay for ⁇ -galactosidase activity is described in Breeden, et al, Cold Spring Harb. Symp. Quant. Biol., 1985, 50, 643).
  • Positive AD-library plasmids are rescued from transformants and reintroduced into the original yeast strain as well as other strains containing unrelated DNA-BD fusion proteins to confirm specific voltage-gated ion channel polypeptide/library protein interactions.
  • Insert DNA is sequenced to verify the presence of an open reading frame fused to GAL4 AD and to determine the identity of the voltage-gated ion channel polypeptide-binding protein.
  • FRET fluorescence resonance energy transfer
  • a fusion of the nucleotide sequences encoding an intracelllular soluble portion of the voltage-gated ion channel polypeptide and CFP is constructed in an appropriate plasmid, using standard subcloning techniques.
  • a nucleotide encoding a YFP fusion of the possibly interacting target protein is constructed in a second plasmid.
  • the CFP/voltage-gated ion channel polypeptide fusion construct is verified by sequencing. Similar controls are performed with the YFP/target protein construct.
  • the expression of each protein can be monitored using fluorescence techniques (e.g., fluorescence microscopy or fluorescence spectroscopy).
  • Host cells are transformed with both the CFP/voltage-gated ion channel polypeptide and YFP/target protein fusion plasmids according to standard procedure.
  • In situ interactions between CFP/ voltage-gated ion channel polypeptide and the YFP/target protein are detected by monitoring the YFP fluorescence after exciting the CFP fluorophore. The fluorescence is monitored using fluorescence microscopy or fluorescence spectroscopy.
  • changes in the interaction due to e.g., external stimuli are measured using time-resolved fluorescence techniques.
  • a YFP fusion library may be constructed from cDNA of potential voltage- gated ion channel polypeptide-binding proteins.
  • Host cells are transformed with both the CFP/voltage-gated ion channel polypeptide and YFP fusion library plasmids. Clones exhibiting FRET are then isolated and the protein interacting with a voltage-gated ion channel polypeptide is identified by rescuing and sequencing the DNA encoding the YFP/target fusion protein.
  • FLIPR Fluorometric Imaging Plate Reader
  • HTS cell-based, l igh-throughput screening
  • a cell-membrane permeable fluorescent indicator dye suitable for measuring changes in membrane potential such as diBAC (bis-(l,3-dibutylbarbituric acid)pentamethine oxonol, Molecular Probes).
  • diBAC bis-(l,3-dibutylbarbituric acid)pentamethine oxonol, Molecular Probes.
  • COS cells that have been transfected with an ion channel gene of interest are bathed in diBAC. Due to the presence of both endogenous potassium channels in the cells as well as the transfected channel, the addition of 30 mM extracellular potassium causes a membrane depolarization. This results in an increase in diBAC uptake by the cell, and thus an overall increase in fluorescence.
  • a potassium channel opener such as chromakalim
  • the membrane is hyperpolarized causing a net outflow of diBAC, and thus a reduction in fluorescence. In this manner the effect of unknown test compounds on membrane potential can be assessed using this assay.

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Abstract

La présente invention concerne le gène KCNA7 mammalien codant pour une protéine du canal potassique potentiel-dépendant. L'invention concerne également des procédés d'identification d'agents capables de moduler l'activité du canal potassique potentiel-dépendant et l'utilisation de ces agents dans le traitement des états liés au canal ionique, par exemple la schizophrénie, la dépression, les accidents vasculaires cérébraux, la migraine, l'épilepsie, le diabète et les maladies neurodégénératives. Les agents bloquant le canal ionique KCNA7 sont censés, entre autres, augmenter la libération d'insuline et, ce faisant, réduire l'hyperglycémie associée au diabète non insulino-dépendant.
PCT/SE2001/002552 2000-11-17 2001-11-16 Sequences nucleotidiques et polypeptidiques de kcna7, une proteine mammalienne du canal potassique potentiel-dependant, et utilisations de ces dernieres WO2002040534A1 (fr)

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SE0004228-3 2000-11-17
SE0004228A SE0004228D0 (sv) 2000-11-17 2000-11-17 Nucleotide sequences
SE0100742A SE0100742D0 (sv) 2001-03-06 2001-03-06 Nucleotide sequences II
SE0100742-6 2001-03-06

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010015421A1 (fr) * 2008-08-08 2010-02-11 Max-Planck-Gesellschaft zur Förderung der Wissenschaften e.V. Berlin Utilisation de la conkunitzine-s1 pour la modulation des sécrétions d'insuline induites par le glucose

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5559009A (en) * 1994-03-04 1996-09-24 The Regents Of The University Of California Voltage-gated potassium channel gene, KV1.7, vectors and host cells comprising the same, and recombinant methods of making potassium channel proteins
US6083986A (en) * 1996-07-26 2000-07-04 Icagen, Inc. Potassium channel inhibitors
US6147098A (en) * 1998-05-11 2000-11-14 Novo Nordisk A/S Substituted guanidines and diaminonitroethenes, their preparation and use

Patent Citations (3)

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Publication number Priority date Publication date Assignee Title
US5559009A (en) * 1994-03-04 1996-09-24 The Regents Of The University Of California Voltage-gated potassium channel gene, KV1.7, vectors and host cells comprising the same, and recombinant methods of making potassium channel proteins
US6083986A (en) * 1996-07-26 2000-07-04 Icagen, Inc. Potassium channel inhibitors
US6147098A (en) * 1998-05-11 2000-11-14 Novo Nordisk A/S Substituted guanidines and diaminonitroethenes, their preparation and use

Non-Patent Citations (2)

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Title
KATALIN KALMAN ET AL.: "Genomic organization, chromosomal localization, tissue distribution and biophysical characterization of a novel mammalian shaker-related voltage-gated potassium channel, Kv1.7", JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 273, no. 10, March 1998 (1998-03-01), pages 5851 - 5857, XP002908135 *
VLADIMIR I. KASHUBA ET AL.: "Initial isolation and analysis of the human Kv1.7 (KCNA7) gene, a member of the voltage-gated potassium channel gene family", GENE, vol. 268, 2001, pages 115 - 122, XP004240569 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010015421A1 (fr) * 2008-08-08 2010-02-11 Max-Planck-Gesellschaft zur Förderung der Wissenschaften e.V. Berlin Utilisation de la conkunitzine-s1 pour la modulation des sécrétions d'insuline induites par le glucose
EP2154152A1 (fr) * 2008-08-08 2010-02-17 Max-Planck-Gesellschaft zur Förderung der Wissenschaften e.V. Utilisation de Conkunitzin-S1 pour la modulation de sécrétion d'insuline induite par glucose

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