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WO2001068698A2 - Nouveau canal cationique non selectif - Google Patents

Nouveau canal cationique non selectif Download PDF

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Publication number
WO2001068698A2
WO2001068698A2 PCT/EP2001/002837 EP0102837W WO0168698A2 WO 2001068698 A2 WO2001068698 A2 WO 2001068698A2 EP 0102837 W EP0102837 W EP 0102837W WO 0168698 A2 WO0168698 A2 WO 0168698A2
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WIPO (PCT)
Prior art keywords
nucleic acid
otrpc4
sequence
polypeptide
host
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PCT/EP2001/002837
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German (de)
English (en)
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WO2001068698A3 (fr
Inventor
Guenter Schultz
Timothy Plant
Rainer Strotmann
Christian Harteneck
Karin Nunnenmacher
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Boehringer Ingelheim Pharma Kg
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Publication of WO2001068698A2 publication Critical patent/WO2001068698A2/fr
Publication of WO2001068698A3 publication Critical patent/WO2001068698A3/fr

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    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • 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
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals

Definitions

  • the present invention relates to nucleic acids which code for the non-selective cation channel OTRPC4 and to polypeptides which are coded by said nucleic acids.
  • the invention further relates to hosts or host cells, said
  • the invention includes • blockers, activators and modulators of said OTRPC4 cation channels and pharmaceutical compositions containing said blockers, activators and modulators.
  • the invention also relates to non-human mammals which contain either OTRPC4 as a transgene, inactivated gene (knock-out) or modified gene (knock-in).
  • RVD regulated volume decrease
  • TRP transient receptor potential
  • This channel is for the C. elegans reactions to solutions with high Responsible for osmolarity and was therefore called OSM-9 (2), but so far nothing is known about the biophysical characteristics of OSM-9, and a corresponding homologous protein has not yet been described for mammals.
  • the family of TRP channels (TRPCs) (3) can be divided into three different subfamilies (4).
  • the largest family is the STRP subfamily (short TRP; named after its short N-terminus), consisting of the classic Drosophila channels TRP and TRPL (transient receptor potential-like) (5) and 7 mammalian homologues from TRP (TRPC1- 7 ) (6-15).
  • the channels of this family are involved in the calcium influx that is triggered by the activation of receptors that have in common that. they activate the phospholipase C.
  • the second subfamily of TRPCs was named OTRPC, after the first representative of this family OSM-9. The channels of this family are activated by chemical and physical stimuli.
  • the OTRPC family includes the vanilloid receptor (VRl) (16, 17), the vanilloid-like receptor (VRL-1, also known as GRC) (18, 19), and a channel whose possible function is that of an epithelial Calcium channel is (ECaC or also known as CaTl) (20, 21).
  • VRl is a non-selective calcium permeable channel that was cloned from rat dorsal ganglion cells (16). This channel is activated by heat and the substance capsaicin, which causes pain.
  • the recently criticized, VRR-related channel, VRL-1 can be activated by heat and could be involved in pain reception (18).
  • IGF-1 insulin-like growth factor-1
  • ECaC ECaC
  • CaTl 21
  • LTRPC long TRP channels, named after its long N-terminus
  • WO 00/32766 discloses human vanilloid receptors and their use.
  • the object of the present invention is to provide a new TRP channel with advantageous properties compared to the channels described above and known from the prior art. Description of the invention
  • RNA and RNA or DNA and DNA each have the same meaning.
  • the invention relates to a nucleic acid, characterized in that it codes for the non-selective cation channel OTRPC4 or for a fragment, a functional variant, an allele variant, a subunit, or variants of the said nucleic acid due to the degenerative code or a nucleic acid which is linked to said nucleic acid can hybridize.
  • the cation channel or OTRPC4 polypeptides according to the invention are described further below.
  • OTRPC4 nucleic acids according to the invention are preferably eukaryotic nucleic acids, particularly preferably human or murine, but also from rats, hamsters, goats, cattle, pigs, sheep, dogs, cats, monkeys and other eukaryotes known to the person skilled in the art.
  • said nucleic acid is a recombinantly produced nucleic acid, e.g. a cDNA.
  • nucleic acids according to the invention are shown as examples.
  • a nucleic acid RNS according to the invention is preferred.
  • a nucleic acid DNA according to the invention is further preferred.
  • a nucleic acid according to the invention is furthermore preferably characterized in that it contains 5 'or 3' or 5 'and 3' untranslated regions.
  • the nucleic acid according to the invention can contain further untranslated regions upstream and / or downstream.
  • Said untranslated region can comprise a regulatory element, such as a transcription initiation unit (promoter) or enhancer.
  • Said promoter can be, for example, a structurally active or inducible or development-controlled promoter.
  • Preferred, without excluding other known promoters are the constitutive promoters of human cytomegalovirus (CMV) and rous sarcoma virus (RSV), as well as simian virus 40 (SV40) and herpes simplex virus (HSV) promoters.
  • CMV cytomegalovirus
  • RSV40 simian virus 40
  • HSV herpes simplex virus
  • Inducible promoters include antibiotic-resistant promoters, heat shock promoters, hormone-inducible "Mouse Mammary Tumor Virus” (MMTV) promoters and the metallothionein promoter.
  • a nucleic acid according to the invention is furthermore preferably characterized in that it codes for a fragment of the non-selective cation channel OTRPC4.
  • a nucleic acid according to the invention is furthermore preferably characterized in that it codes for a functional variant of the non-selective cation channel OTRPC4.
  • a nucleic acid according to the invention is furthermore preferably characterized in that it codes for an allelic variant of the non-selective cation channel OTRPC4.
  • a nucleic acid according to the invention is furthermore preferably characterized in that it codes for variants of the nucleic acid on the basis of the degenerative code.
  • a nucleic acid is furthermore preferably characterized in that it can hybridize to a nucleic acid according to the invention under stringent conditions. Stringent conditions are known to the person skilled in the art and can be found in particular in Sambrook et al. (1989). Molecular Cloning: A Laboratory Manual, 2nd ed, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York..
  • a nucleic acid according to the invention is furthermore preferably characterized in that
  • said non-selective cation channel OTRPC4 is a mammalian cation channel.
  • a nucleic acid according to the invention is furthermore preferably characterized in that said non-selective cation channel OTRPC4 is murine.
  • a nucleic acid according to the invention is furthermore preferably characterized in that said non-selective cation channel OTRPC4 is human. Also preferred is a nucleic acid which is characterized in that it has the sequence
  • R is understood as A or G
  • M as A or C
  • S as i C or G
  • Y as C or T
  • K as G or T
  • W as A or T.
  • nucleic acid which is characterized in that the sequence CTCTCACCGCCTACTACCAGCCGCTGGAGGGCACAATGGCGGATTCCAGCGAA w GGCCCCCGCGCGGGGCCCGGGGAGGTGGCTGAGCTCCCCGGGGATGAGAGTGG CACCCCAGGTGGGGAGGCTTTTCCTCTCCTCCCTGGCCAATCTGTTTGAGGG GGAGGATGGCTCCCTTTCGCCCTCACCGGCTGATGCCAGTCGCCCTGCTGGCCC AGGCGATCrGGCGACCAAATCTGCGCATGAAGTTCCAGGGCGCCTTCCGCAAGG GGGTGCCCAACCCCATCGATCTGCTGGAGTCCACCCTATATGAGTCCTCGGTGG TGCCTGGGCCCAAGAAAGCACCCATGGACTCACTGTTTGACTACGGCACCTATC GTCACCACTCCAGTGACAACAAGAGGTGGAGGAAGAAGATCATAGAAGCAG CCGCAGAGCCCCAAAGCCTGCCTCAGCCGCCCCATCCTCAAAG
  • Conditions can hybridize, an allelic variant or a functional variant of said sequence or a variant of the nucleic acid comprises due to the degenerative code.
  • the human OTRPC4 cDNA sequence is included with the sequence shown above.
  • nucleic acid which is characterized in 'that the
  • Can hybridize conditions comprises an allelic variant or a functional variant of said sequence or a variant of the nucleic acid based on the degenerative code, wherein R is an A or G, M is an A or C, S is a C or G, Y is a C or T, K can be a G or T and W can be an A or T. According to the invention is shown with the above
  • Sequence comprises the murine OTRPC4 DNA sequence with 5 'and 3' untranslated sequences.
  • R is A or G
  • M is A or C
  • S is C or G
  • Y is C or T
  • K is G or
  • T and W can be an A or T.
  • the sequence shown above is the murine OTRPC4 DNA sequence with 5 'and 3' untranslated sequences.
  • Conditions can hybridize, an allelic variant or a functional variant of said sequence or a variant of the nucleic acid comprises due to the degenerative code.
  • the murine OTRPC4 cDNA sequence is included with the sequence shown above.
  • a recombinant vector is characterized in that it contains a nucleic acid according to the invention, as described above.
  • vectors according to the invention are viral vectors such as e.g. B. Vaccinia, Semliki Forest Virus and Adenovirus.
  • Vectors for use in COS cells have the Simian Virus (SV) 40 “origin of replication” and enable high numbers of copies of the plasmids.
  • Vectors for use in insect cells are, for example, E.
  • coli transfer vectors and contain, for example, the DNA coding for polyhedrin as a promoter
  • a recombinant vector according to the invention is characterized in that it is an expression vector.
  • a host characterized in that it contains a vector according to the invention.
  • a host according to the invention expresses an OTRPC4 polypeptide according to the invention, for example on the cell surface
  • the hosts according to the invention can be transiently or stably transfected with one of the said vectors, such a host is described by way of example in Example 1, Figure 4 of the invention.
  • Eukaryotic host cells according to the invention include fungi, such as. B. Pichia pastoris, Saccharomyces cerevisiae, Schizosaccharomyces, Trichoderma.
  • Another preferred host according to the invention is an insect cell (e.g. from Spodoptera frugiperda Sf-9, with a baculovirus expression system).
  • Cells according to the invention also include oocytes, for example from frogs or toads.
  • Hosts according to the invention can also be plant cells, e.g. by Nicotiana tabacum.
  • the OTRPC4 polypeptides according to the invention are particularly well expressed in mammalian cells and cell lines. Therefore, a preferred host according to the invention is a mammalian cell. Examples of mammalian cells according to the invention are HEK293, HeLa, COS, BHK, CHO cells.
  • a host according to the invention is therefore very particularly preferably an Sf9, HEK293 or HeLa cell.
  • a host according to the invention is preferably a bacteriophage. Baculovirus may be mentioned as an example.
  • Another host according to the invention is a prokaryotic host cell. Examples of prokaryotic host cells according to the invention are Escherichia coli, Bacillus subtilis, Streptomyces or Proteus mirabilis.
  • Another important aspect of the present invention relates to a polypeptide which is encoded by a nucleic acid according to the invention or a fragment, a functional variant, an allele variant, a subunit, a variant based on the degenerative nucleic acid code or a glycosylation variant thereof.
  • OTRPC4 polypeptide or fragment thereof is understood to mean one or more of the polypeptide (s) described here, ie a polypeptide selected from fragments, allelic variants, functional subunits, variants based on the degenerative nucleic acid code, a chemical derivative thereof, a fusion protein with said polypeptide or a glycosylation variant of OTRPC4.
  • OTRPC4 polypeptides according to the invention are preferably eukaryotic polypeptides, particularly preferably human or murine, but also from rats, hamsters, goats, cattle, pigs, sheep, dogs, cats, monkeys and from other eukaryotes known to the person skilled in the art.
  • OTRPC4 in the context of this invention is a new cation channel which, compared to the cation channels known from the prior art, has the advantageous property that it is regulated by changes in the osmolarity of the extracellular medium. It therefore represents a completely new generation of cation channels compared to known from the prior art cation channels, which are responsible, for example, as osmosensors for the regulation of the cell volume.
  • the channel activity is stimulated and inhibited by increasing it.
  • the channel is constitutively active with a physiological osmolarity of approx. 300 mosmol / l.
  • the channel is non-selective in its ion permeability, that is, it is continuous for all cations (Na + , K + , Ca 2+ ) and shows, for example, a certain preference for Ca 2+ ca ->) •
  • polypeptide according to the invention which is a fragment of the non-selective cation channel OTRPC4. This means part of the polypeptide according to the invention.
  • polypeptide according to the invention which is a functional variant of the non-selective cation channel OTRPC4.
  • OTRPC4 can differ from OTRPC4 by substitution, deletion or addition of one or more amino acids, preferably by 1 to 10 amino acids.
  • functional variants are understood to mean further representatives of the OTRPC4 family, which likewise have the above-described advantageous property of regulating channel activity by osmolarity.
  • Another important aspect of the present invention relates to a polypeptide according to the invention, which is an allelic variant of the non-selective cation channel OTRPC4.
  • polypeptide according to the invention which is a subunit of the non-selective cation channel OTRPC4.
  • Ion channels are often composed of subunits, for example the AMPA receptor. Accordingly, the invention also encompasses subunits of the OTRPC4 cation channel.
  • Another important aspect of the present invention relates to a polypeptide according to the invention, which is a variant of the non-selective cation channel OTRPC4 due to the degenerative nucleic acid code.
  • polypeptide according to the invention which is a chemical derivative of the non-selective cation channel OTRPC4.
  • OTRPC4 a chemical derivative of the non-selective cation channel
  • This is understood to mean molecules which are produced from the OTRPC4 polypeptides according to the invention by chemical reactions, for example by iodination, acetylation, binding to an effector molecule or radioisotope or to a toxin.
  • a polypeptide according to the invention which is a fusion protein from the non-selective cation channel OTRPC4 and another protein.
  • a fusion protein can be produced, for example, by recombinant expression of the OTRPC4 nucleic acid according to the invention, which is fused to a further nucleic acid which contains all the coding information “in frarne”.
  • This can be, for example, a marker protein or a reporter protein such as GFP or LacZ.
  • Other fusion partners are known to the person skilled in the art.
  • Another important aspect of the present invention relates to a polypeptide according to the invention, which is a glycosylation variant of the non-selective cation channel OTRPC4.
  • the invention comprises processes for the production of polypeptides according to the invention, characterized in that a host according to the invention is cultivated and said polypeptide is expressed.
  • Said hosts can be transfected, for example, stably or transiently with a vector or an expression vector which contains a nucleic acid coding for an OTRPC4 polypeptide or fragment.
  • the OTRPC4 polypeptide or fragment according to the invention is expressed on the cell surface of the host.
  • Said polypeptide can also be secreted into the medium.
  • the OTRPC4 polypeptides or fragments according to the invention can be used in a method according to the invention, for example in fungi, such as. B.
  • Pichia pastoris Saccharomyces cerevisiae, Schizosaccharomyces, Trichoderma with vectors that lead to surface expression, are prepared.
  • the method according to the invention for producing the OTRPC4 polypeptides or fragments can also be carried out with insect cells, eg. B. as a transient or stable expression system or baculovirus expression system.
  • insect cells eg. B. as a transient or stable expression system or baculovirus expression system.
  • Sf-9 insect cells are infected with, for example, Autographa californica nuclear polyhedrosis virus (AcNPV) or related viruses.
  • AcNPV Autographa californica nuclear polyhedrosis virus
  • the E. coli transfer vectors described above contain, for example, as a promoter the DNA coding for polyhedrin, behind which the DNA coding for the OTRPC4 polypeptide or fragment according to the invention is cloned.
  • Insect cell expression systems for the expression of OTRPC4 polypeptide or fragment are commercially available.
  • a mammalian expression system for example in a host according to the invention, for example the HEK293 cell or the Heia cell, which contains, for example in an expression vector, a nucleic acid according to the invention coding for OTRPC4 or a fragment thereof, can be used to express the OTRPC4 cation channel, the said Host is cultivated under conditions known to those skilled in the art and the OTRPC4 polypeptide or fragment is expressed, for example, on the cell surface.
  • An advantage of mammalian expression systems is that they enable very good glycosylation and folding conditions. Mammalian cells are with transient expression systems, stable expression systems and with viral expression systems such.
  • B. Vaccinia, Semliki Forest virus, adenovirus can be used, which are commercially available.
  • transgenic animals e.g. B. cows, goats, mice are suitable for a method according to the invention.
  • Transgenic plants such as Nicotiana tabacum (tobacco) can also be used in a method according to the invention. These are particularly suitable for the production of OTRPC4 polypeptide or fragment according to the invention.
  • OTRPC4 fragments according to the invention are preferably suitable for OTRPC4 fragments according to the invention, but also for the entire OTRPC4 polypeptide.
  • the OTRPC4 polypeptides according to the invention are either preferably on the surface, for example in the outer shell, i.e. one of the two bacterial cell membranes or the pyptidoglycan layer of the outer shell in Gram-negative bacteria or integrated in the cell membrane in Gram-positive bacteria, or intracellularly, e.g. in inclusion bodies or by periplasmic secretion in Gram-negative bacteria using vectors suitable for this purpose.
  • Another aspect of the present invention relates to an antibody protein, characterized in that it is specific for a polypeptide according to the invention.
  • the antibody protein according to the invention therefore binds to an epitope of OTRPC4 or to an epitope of one of the variants described above.
  • fragment antigen-binding Fab
  • These OTRPC4-specific antibody proteins according to the invention consist of the variable regions of both Chains that are held together by the subsequent constant region. These can arise from conventional antibodies by digestion of proteases, such as with papain, but similar Fab fragments can now also be produced using genetic engineering.
  • an antibody protein according to the invention is an F (ab ') 2 fragment which can be produced by proteolytic cleavage with pepsin.
  • an OTRPC4-specific antibody molecule according to the invention is such an Fv fragment. Because these Fv fragments are the covalent linkage of both chains because the cysteines of the constant chains are missing, the Fv fragments are often stabilized.
  • an antibody protein is referred to as an Fv single chain or "single-chain Fv" (scFv).
  • scFv single chain Fv
  • Prior art examples of such scFv antibody proteins are described in Huston et al. (1988, PNAS 16: 5879-5883). Therefore, in yet another preferred embodiment, an OTRPC4-specific antibody protein according to the invention is an Fv single chain protein (scFv).
  • an antibody protein according to the invention is a diabody specific for an OTRPC4 epitope.
  • Antibody fragment The person skilled in the art understands diabody as a bivalent homodimeric scFv derivative (Hu et al., 1996, PNAS 16: 5879-5883). The shortening of the linker in an scFv molecule to 5-10 amino acids leads to the formation of homodimers in which an inter-chain VH / VL assembly takes place. Diabodies can also be stabilized by incorporating disulfide bridges. Prior art examples of diabody antibody proteins are found in Perisic et al. (1994, Structure 2: 1217-1226).
  • minibody as a bivalent, homodimeric scFv derivative. It consists of a fusion protein which contains the CH3 region of an immunoglobulin, preferably IgG, very particularly preferably IgGl as the dimerization region, which is linked to the scFv via an Hwge region (eg also from IgGl) and an E / ' rcfer region is.
  • the disulfide bridges in the hinge region are mostly formed in higher cells and not in prokaryotes.
  • an antibody protein according to the invention is an OTRPC4-specific minibody antibody fragment.
  • Prior art examples of minibody antibody proteins can be found in ⁇ u et al. (1996, Cancer Res. 56: 3055-61).
  • triabody as: a trivalent homotrimeric scFv derivative (Kortt et al. 1997 Protein Engineering 10: 423-433). ScFv derivatives in which V ⁇ -VL are fused directly without a linker sequence lead to the formation of trimers.
  • a tetravalent miniantibody as a tetravalent homodimeric scFv derivative (Pack et al., 1995 J. Mol. Biol. 246: 28-34). The multimerization is carried out by tetrameric coiled c ⁇ zY domain.
  • An antibody protein according to the invention is particularly preferably completely human.
  • Another aspect of the present invention relates to a method for producing an antibody protein according to the invention, which comprises the following steps: a host selected from a eukaryotic or prokaryotic cell which contains one or more vectors with one or more nucleic acids specific for the antibody protein, is cultured under conditions in which said antibody protein is expressed by said host cell, and said antibody protein is isolated.
  • the antibody proteins of the invention can also be used in a method according to the invention in fungi such as. B. Pichia pastoris, Saccharomyces cerevisiae, Schizosaccharomyces, Trichoderma with vectors which lead to intracellular expression or secretion, getting produced.
  • the method according to the invention for producing the antibody proteins can also be carried out with insect cells, for. B. as a transient or stable expression system or baculovirus expression system, comparable to that described above.
  • Insect cell expression systems for the expression of antibody proteins are commercially available. Insect cell expression systems are particularly suitable for the scFv fragments according to the invention and Fab or F (ab ') 2 fragments and antibody proteins or fragments thereof, which are fused with effector molecules, but also for complete antibody molecules.
  • mammalian expression systems allow very good glycosylation and folding conditions, e.g. transient expression systems, e.g. B. in COS cells or stable expression systems such. B. BHK, CHO, myeloma cells. Mammalian cells are also e.g. B. with viral expression systems such. B. Vaccinia, Semliki Forest virus, adenovirus can be used. Also transgenic animals e.g. B. cows, goats, mice are suitable for a method according to the invention. Transgenic plants such as Nicotiana tabacum (tobacco) can also be used in a method according to the invention.
  • transient expression systems e.g. B. in COS cells or stable expression systems such.
  • Mammalian cells are also e.g. B. with viral expression systems such. B. Vaccinia, Semliki Forest virus, adenovirus can be used.
  • transgenic animals e.
  • the secretion of the antibody protein into the interstitial space can be achieved.
  • Production with prokaryotic expression systems such as Escherichia coli, Bacillus subtilis, Streptomyces or Proteus mirabilis is preferably suitable for antibody fragments according to the invention, such as Fab, F (ab ') 2, scFv fragments, minibodies, diabodies and multimers of said fragments.
  • the antibody proteins according to the invention are either intracellular, e.g. in inclusion bodies or by periplasmic secretion in Gram-negative bacteria using suitable vectors.
  • the invention also encompasses the use of a polypeptide according to the invention for finding blockers, activators or modulators of said polypeptides.
  • Blockers are understood to mean substances which inhibit the ion permeability of the channel by binding to the OTRPC4 cation channel itself, by binding to regulatory subunits or by interaction with the cell membrane or parts thereof.
  • Activators are understood to mean substances which stimulate the ion permeability of the channel by binding to the OTRPC4 cation channel itself, by binding to regulatory subunits or by interaction with the cell membrane or parts thereof.
  • Modulators are understood to mean substances which, by binding to the OTRPC4 cation channel itself, by binding to regulatory subunits or by interaction with the cell membrane or parts thereof, change the ion permeability of the channel, for example change the selectivity of the channel towards calcium and sodium.
  • Blockers, activators or modulators can develop their respective pharmacological properties depending on physical influences, such as pH, temperature and ion concentrations of the intracellular or extracellular mileus, or also depending on the activation state of the channel.
  • the invention furthermore encompasses the use of a host according to the invention for finding blockers, activators or modulators of OTRPC4 channels.
  • Another preferred aspect of the invention is a method for finding blockers, activators or modulators of OTRPC4, characterized in that a host according to the invention is incubated with a test substance.
  • a further, very particularly preferred aspect of the invention is a method according to the invention, characterized in that a membrane flow is measured, said membrane flow is compared with a membrane flow which is measured in said host after incubation with a known control substance or in the absence of the test substance.
  • a membrane flow is measured, said membrane flow is compared with a membrane flow which is measured in said host after incubation with a known control substance or in the absence of the test substance.
  • a method is described by way of example in Example 1, Figure 7 of the invention.
  • Another, very particularly preferred aspect of the invention is a method according to the invention in which said activator is bound to a channel, said host is incubated with a test substance and the displacement of the activator bound to the channel is measured by the test substance.
  • a further, very particularly preferred aspect of the invention is a method according to the invention, in which a host according to the invention is incubated with a test substance, the intracellular amount of a divalent cation is determined and said amount of the divalent cation is compared with the amount of said divalent cation, which in the Incubation of said hosts with a known control or in the absence of the Test substance is measured.
  • a method is described by way of example in Example 1 of the invention.
  • HTS refers in the context of the invention to an experimental method in which a large number of test substances are tested simultaneously, preferably an HTS method is carried out in microtiter plates, partially or completely automated and to electronic devices such as computers for data storage, analysis and interpretation by means of bioinformatics Robots which can handle a large number of microtiter plates at the same time and can carry out several thousand tests per day are preferably used for automation purposes, and a test substance is preferably tuned to a desired activator, blocker or modulator radio tion tested in a line-based system with a cell according to the invention.
  • the term HTS also includes ultra high throughput patterning (UHTS) tests.
  • UHTS ultra high throughput patterning
  • said UHTS methods are performed using 384 or 1536 well microtiter plates, submicroliter and subnanoliter pipettors, improved plate readers, and methods to prevent evaporation.
  • HTS processes are described by way of example in the patents US 5876946 A or US 5902732 A. The average person skilled in the art can adapt the methods described above and in the examples to an HTS or UHTS format without being inventive.
  • a HTS for identifying blockers, activators or modulators of the OTRPC4 channel can be carried out as described in Example 1, but can also be carried out using so-called inducible expression systems, for example a plasmid inducible by tetracycline (Gossen M, Bonin AL, Freundlich S, Bujard H: Inducible gene expression Systems for higher eukaryotic cells. Curr Opin Biotechnol 1994, 5, 516-20) or a system inducible by the ecdysone receptor (Invitrogen). These systems, as well as others, are commercially available.
  • Another important aspect of the present invention is an activator of OTRPC4, which can be found using a method according to the invention.
  • Another important aspect of the present invention is a blocker of OTRPC4, which can be found using a method according to the invention.
  • a modulator of OTRPC4 is a modulator of OTRPC4, which can be found using a method according to the invention.
  • a further preferred embodiment of the invention comprises an anti-sense nucleic acid, characterized in that it can hybridize to a part of a nucleic acid according to the invention under stringent conditions.
  • Anti-sense nucleic acid (English: “anti-sense nucleic acid” or also “anti-sense oligonucleotides”) in the context of this invention is understood to mean DNA or RNA molecules which are complementary to at least part of an inventive, i.e. mRNA coding for an OTRPC4 polypeptide or fragment.
  • a definition of anti-sense nucleic acid can also be found in the prior art (Weintraub HM, 1990 Scientific American, 262, 34-40). In the cell, anti-sense nucleic acid molecules hybridize to the corresponding mRNA and form a double-stranded molecule.
  • the anti-sense nucleic acids according to the invention interfere with the translation of the mRNA coding for OTRPC4 polypeptide or an OTRPC4 fragment, since the cell will not translate said double-stranded mRNA.
  • the central region of the anti-sense nucleic acid in the context of this invention contains at least 14 nucleotides that are complementary to the target RNA.
  • the invention also encompasses peptide nucleic acids, phosphodiester anti-sense nucleic acids and phosphothioate oligonucleotides which are complementary to at least part of an mRNA molecule coding for an OTRPC4 polypeptide or fragment according to the invention.
  • Such substances specific for other target RNA are known from the prior art (Boado RJ et al., 1998 JPharm Sei 87: 1308-1315.).
  • Example 1 In Example 1 (Table 1), five anti-sense sequences are listed as examples and the criteria that led to the selection of these sequences are shown.
  • Another preferred embodiment of the invention comprises an anti-sense nucleic acid according to the invention which is a ribozyme.
  • Ribozyme in the context of this invention is an RNA molecule that is specific to the target RNA, i.e. the invention for a
  • the ribozyme according to the invention preferably has a central sequence which is not complementary to and for the target RNA catalytic activity (catalytic region (a)) and two flanking sequences which are essentially complementary to two adjacent sequences of the target RNA (hybridization region (b)), so the binding of the ribozyme via base pairing and thereby the selective cleavage of the target Allow RNS.
  • a preferred embodiment of the ribozyme according to the invention can be represented by the following general formula: (b) (a) (b)
  • N G, C, A or U
  • R is purine and S is pyrimidine and the central region is No -3 o Sequence (a) can be replaced by a linker that is not a nucleic acid, namely, for example, a hydrocarbon chain (see also Thomson et al., 1993, Nucleic Acids Res 21, 5600-5603.).
  • the ribozymes according to the invention can be, for example, a "hammerhead""hairpin” or "axehead” ribozyme.
  • ribozyme can also be modified within the scope of the invention to obtain increased nuclease resistance, examples of which are the substitution of the 2'-OH groups of the ribose by 2'-H, 2'-O-methyl, 2'-O-allyl, 2'-fluorine or 2'- Amino groups (Paolella et al., 1992, and Pieken et al., 1991) or the modification of phosphodiester bonds, e.g.
  • RNA Ribonucleic acid
  • pharmaceutically acceptable carriers or auxiliaries phosphorothioate or phosphorodithioate
  • Eckstein, 1985, and Beaton et al. In : Eckstein, F. (ed.) Oligon ucleotides and analogues - A practical approach - Oxford, JRL Press (1991), 109-135) or by a methyl group (methylphosphonate; Miller, ibid., 137-154).
  • Further modifications include the conjugation of the RNA with poly-L-lysine, polyalkyl derivatives, cholesterol or PEG.
  • the ribozymes according to the invention preferably contain at least one of the phosphate modifications described above and / or at least one of the ribose modifications described above.
  • Another important aspect of the invention is a pharmaceutical composition which contains a nucleic acid according to the invention and pharmaceutically acceptable carriers or auxiliaries.
  • compositions which contains an anti-sense nucleic acid according to the invention and pharmaceutically acceptable carriers or excipients.
  • compositions which contains a polypeptide according to the invention and pharmaceutically acceptable carriers or excipients.
  • Pharmaceutically acceptable carriers or adjuvants in this invention can be physiologically acceptable compounds that, for example, stabilize or improve the absorption of OTRPC4 activator, blocker or modulator.
  • physiologically acceptable compounds include, for example, carbohydrates such as glucose, sucrose or dextrans, antioxidants such as ascorbic acid or glutathione, chelating agents, low molecular weight compounds or other stabilizers or auxiliaries (see also Remington's Pharmaceutical Sciences, 18th edition, Mack Publ., Easton.).
  • Those skilled in the art know that the selection of a pharmaceutically acceptable carrier e.g. depends on the administration route of the connection.
  • compositions which contains a vector according to the invention and pharmaceutically acceptable carriers or excipients.
  • Said pharmaceutical composition can also contain a vector according to the invention for gene therapy and can additionally comprise as an adjuvant a colloidal dispersion system or liposomes for targeted application of the pharmaceutical composition.
  • a pharmaceutical composition which contains a host according to the invention and pharmaceutically acceptable carriers or auxiliaries. Also a host or a host cell which has a vector according to the invention contains, can be used in a pharmaceutical composition in the context of this invention, for example for gene therapy.
  • colloidal dispersion system An example of a targeted application system, e.g. for anti-sense oligonucleotides or ribozymes according to the invention is said colloidal dispersion system.
  • Colloidal dispersion systems include macromolecule complexes, nanocapsules, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles and lipomas, or liposome formulations.
  • the preferred colloidal system of the invention is liposomes. Liposomes are artificial membrane vesicles that are useful as vehicles in vitro and in vivo. These formulations can carry cationic, anionic or neutral charge.
  • RNA, DNA and intact virions can enter the aqueous phase in the Encapsulated internally and transported to the target in a biologically active form (Fraley R et al., 1981, Trends s Biochem Sei 6, 77-80).
  • liposomes In addition to mammalian cells, liposomes have also been used for the targeted transport of nucleotides in plant,
  • the genes should be included with high efficiency without reducing their biological activity; (2) it should be preferential and 0 substantial binding to the target cell compared to non-target cells; (3) the aqueous phase of the vehicle should be with ho efficiency can be transferred into the target cell cytoplasm; and (4) the genetic information should be expressed accurately and efficiently (Mannino RJ et al., 1988, BioTechniques 6, 682-690).
  • composition of the liposomes usually consists of a combination of phospholipids, in particular high-phase transition-temperature phospholipids (English “high-phase transition temperature"), e.g. in combination with steroids, e.g. Cholesterol. Other phospholipids or other lipids can also be used.
  • the physical characteristics of the liposomes depend on the pH, the ion concentration and the presence of divalent cations.
  • the pharmaceutical composition of the present invention can also contain a vector according to the invention as a bare “gene expression vector”.
  • a vector according to the invention is not provided with an adjuvant for targeted application associated (e.g. liposomes, colloidal particles, etc.).
  • an adjuvant for targeted application associated e.g. liposomes, colloidal particles, etc.
  • a fundamental advantage of naked DNA vectors is the lack of an immune response that is caused by the vector itself.
  • the invention also relates to the use of a nucleic acid according to the invention for the manufacture of a medicament for the treatment of a disease selected from the group consisting of diabetes, hyperlipidemia, hyperproteinemia, hypertension, stroke, renal insufficiency, shock and other pathophysiological conditions which are characterized by hyper- and hypoosmolarity ,
  • a disease selected from the group consisting of diabetes, hyperlipidemia, hyperproteinemia, hypertension, stroke, renal insufficiency, shock and other pathophysiological conditions which are characterized by hyper- and hypoosmolarity
  • shock states of different origins such as cardiogenic, metabolic, septic, anaphylactic shock or shock states triggered by burns or polytrauma.
  • shock is understood to be a pathophysiological condition which leads to a generalized, serious reduction in tissue perfusion and consecutive tissue damage.
  • the invention also relates to the use of an anti-sense nucleic acid according to the invention for the manufacture of a medicament for the treatment of a disease selected from the group of diabetes, hyperlipidemia, hyperproteinemia, hypertension, stroke, renal insufficiency and and shock.
  • a disease selected from the group of diabetes, hyperlipidemia, hyperproteinemia, hypertension, stroke, renal insufficiency and and shock.
  • shock states of different origins such as cardiogenic, metabolic, septic, anaphylactic shock or shock states triggered by burns or polytrauma.
  • the invention also relates to the use of a vector according to the invention for the manufacture of a medicament for the treatment of a disease selected from the group of diabetes, hyperlipidemia, hyperproteinemia, hypertension, stroke, renal failure and and shock.
  • the invention also relates to the use of a host according to the invention for the manufacture of a medicament for the treatment of a disease selected from the group of diabetes, hyperlipidemia, hyperproteinemia, hypertension, stroke, renal failure and and shock.
  • shock states of different origins such as cardiogenic, metabolic, septic, anaphylactic shock or shock states triggered by burns or polytrauma.
  • Another essential aspect of the invention is a non-human mammal, characterized in that it contains a nucleic acid (transgene) according to the invention in addition to its genome.
  • transgene a nucleic acid according to the invention in addition to its genome.
  • This is understood to mean a non-human, transgenic mammal which, in addition to its genome, has stably integrated a nucleic acid sequence according to the invention for OTRPC4 or a fragment thereof into part of its body cells (chimera) or into all body cells and expresses the OTRPC4 polypeptide or fragment .
  • the person skilled in the art is aware of transgenic mammals which are transgenic for other sequences (see also Schenkel, L, Spektrum Akad. Verl., 1995).
  • Transgenic mammals according to the invention are, for example, transgenic rodents, such as rats, mice and hamsters, but also goats, cattle, pigs, sheep and other non-human mammals known to the person skilled in the art. Mice are very
  • Another essential aspect of the invention is a non-human mammal, characterized in that a nucleic acid according to the invention is inactivated (gene knock-out) in its genome.
  • a nucleic acid according to the invention is inactivated (gene knock-out) in its genome.
  • This is understood to mean a non-human, so-called knock-out mammal, in whose genome the endogenous nucleic acid sequence corresponding to an inventive nucleic acid sequence coding for OTRPC4 or a fragment thereof is inactivated and in which no or only small amounts of OTRPC4 polypeptide or fragment of which are expressed.
  • Small amounts mean that the expression of OTRPC4 polypeptide or fragment is reduced by at least 50%, preferably more than 50 to 80%, particularly preferably more than 80 to 100% compared to comparable, non-knock-out mammals.
  • the inactivation is often carried out by cloning a reporter sequence, for example the gene for neomycin resistance.
  • Other knock-out mammals in which other sequences are inactivated are known to the person skilled in the art.
  • Knockout mammals according to the invention are, for example, knock-out rodents, such as rats, mice and hamsters, however also goats, cattle, pigs, sheep and other non-human mammals known to those skilled in the art. Mice are very particularly preferred. Methods for producing a knock-out mammal according to the invention are described below. The construction of a recombinant vector for a conditional knock-out is described as an example in Example 1.
  • Another essential aspect of the invention is a non-human mammal, characterized in that a nucleic acid according to the invention is modified (gene knock-in) in its genome.
  • This modification can be carried out by means of homologous recombination of the coding nucleic acid and has the result that, for example, an OTRPC4 polypeptide or fragment thereof is expressed in this mammal with modified properties. This can e.g. by mutation in a small part of the coding nucleic acid.
  • Knock-out mammals according to the invention are, for example, knock-in rodents, such as rats, mice and hamsters, but also goats, cattle, pigs, sheep and other non-human mammals known to the person skilled in the art. Mice are very particularly preferred. Methods for producing a knock-in mammal according to the invention are described below.
  • the non-human transgenic or knock-out or knock-in mammals according to the invention are outstandingly suitable for analyzing the function of the OTRPC4 gene or polypeptide.
  • the mammals according to the invention can each be compared with mammals of the same species or advantageously of the same litter (“littermates”) and the function of the polypeptide according to the invention can thereby be investigated.
  • the invention also includes methods for producing a non-human mammal, characterized in that a ) embryonic stem cells of said non-human mammal are transfected with a vector which contains a nucleic acid according to the invention and a
  • Embryonic stem cells can be obtained by cultivating the inner cell mass of blastocysts and multiplied in tissue culture.
  • the differentiation of the stem cells is prevented by culturing them on fibroblast nutrient cells or by adding leukemia inhibiting factor (LIF) to the culture medium.
  • LIF leukemia inhibiting factor
  • the nucleic acid according to the invention is introduced into ES cells, for example DNA coding for OTRPC4 or a fragment thereof, for example by means of transfection, retrovirus infection or electroporation.
  • ES cells for example DNA coding for OTRPC4 or a fragment thereof, for example by means of transfection, retrovirus infection or electroporation.
  • Such a vector carries, for example, the neomycin gene, which confers resistance to G418.
  • Successfully transfected embryonic stem cells can be identified by adding G418 to the culture medium. Only successfully transfected ES can grow under these conditions.
  • transfected ES are, for example, transferred back into blastocysts and these are transferred into the germ line of a female mammal according to the invention.
  • the mutated cells are integrated into the developing embryo and take part in the development of all tissues.
  • the transgene according to the invention enters the germline.
  • Chimeric animals form which can be characterized, for example, by prior selection of ES cells and recipient blastocysts from animals of different coat colors. Multiple breeding of the chimeric animals gives homozygous animals that express the transgene in each tissue.
  • Another method according to the invention for the production of non-human transgenic mammals comprises the isolation of fertilized egg cells, the microinjection of nucleic acid according to the invention coding for OTRPC4 or a fragment thereof, the implantation of said fertilized egg cells into the germ line of a pseudo-pregnant, female animal, said non-human mammal and the examination of the progeny of said female animal with a male animal of the same type for expression of the transgene.
  • the nucleic acid introduced by microinjection preferably DNA, often integrates at a different location than the comparable endogenous nucleic acid, but is usually expressed in the same way.
  • Said nucleic acid according to the invention can be in the genome in one, but also in numerous, ie 2 to several hundred or thousands of copies be integrated. Details on processes for the production of transgenic, non-human
  • Mammals are known to the person skilled in the art (see also Schenkel, J., Spektrum Akad. Verl., 1995).
  • the invention also includes methods for producing a non-human
  • Mammal characterized in that s d) embryonic stem cells of said non-human mammal with a
  • Transfected vector which contains a nucleic acid that can hybridize to a nucleic acid according to the invention under stringent conditions, and is inactivated by inserting an additional nucleic acid sequence, and a recombination between the genomic DNA of said non-human mammal and the nucleic acid contained in the vector enables e ) Stably transfected stem cells from step d) are isolated and these are transferred into the germ line of a female animal of said non-human mammal. f) the offspring of the said female animal from step e) with a male animal of the same type are animals which are characterized by the nucleic acid
  • Step d) express the encoded polypeptide, analyzed.
  • the endogenous gene which corresponds to or comprises an OTRPC4 nucleic acid sequence according to the invention is inactivated by so-called knock-out
  • said gene is activated and inactivated by homologous recombination.
  • homologous recombination to be methods which make it possible to specifically incorporate nucleic acid, for example DNA, into genes.
  • a criticized copy of the endogenous gene is replaced by a non-functional copy. For example, the inserted copy is interrupted by an inserted copy of one or more antibiotic resistance genes, which leads to inactivation.
  • the sequence for the target gene can be interrupted by the neomycin resistance gene.
  • HSV-tk herpes simplex virus thymidine kinase
  • those cells can be identified in which homologous recombination has taken place.
  • an inactivated copy of a nucleic acid coding for OTRPC4 or a fragment s thereof, cloned into a suitable vector is introduced into embryonic stem cells (as described above) using a suitable method, ie by transfection, retrovirus infection or electroporation.
  • the introduced nucleic acid goes in one part the ES with the corresponding cellular copy of the OTRPC4 gene a homologous recombination and replaces the gene with the introduced nucleic acid according to the invention.
  • those ES in which homologous recombination has taken place can be identified by means of the antibiotic G418 and the antiviral substance ganciclovir.
  • ES, in which homologous recombination has occurred are injected into a blastocyst, which is inserted into the uterus of a female non-human mammal of the same type as the ES.
  • Chimeric animals form which can be characterized, for example, by prior selection of ES cells and recipient blastocysts from animals of different coat colors. Multiple breeding of the chimeric animals gives homozygous animals in which the target gene is completely inactivated in each tissue.
  • the invention also encompasses methods for producing a non-human mammal, characterized in that g) embryonic stem cells of said non-human mammal are transfected with a vector which contains a nucleic acid which can hybridize to a nucleic acid according to the invention under stringent conditions, and is modified by insertion of an additional nucleic acid sequence, and a recombination between the genomic DNA of said non-human mammal and the nucleic acid contained in the vector enables h) stably transfected stem cells from step g) to be isolated and into the germ line a female animal of said non-human Mammals are transferred i) the offspring of the said female animal from step h) with a male animal of the same type are analyzed for animals which express the polypeptide encoded by the nucleic acid from step g).
  • the method for producing knock-out animals is carried out in a manner similar to the method for producing knock-out animals, with the difference that the target gene is not inactivated but modified.
  • OTRPC4-DNA, RNA, protein or channel should in no way be seen as limiting the breadth of the invention, but only serve to illustrate the invention in more detail. Additional OTRPC4 DNA, RNA, proteins or channels are shown in the description.
  • RNA transcript of a length of 3.3 kb was provided everything expressed in the liver, heart, kidney and testis was identified ( Figure 1b).
  • RNA purified from the kidney of a mouse a cDNA s with a length of 3277 bp was cloned using the RACE-PCR method contains an open reading frame of 2616 bp (see sequences according to the invention, supra and claims 19 and 20.)
  • the genomic organization of the murine sequence of OTRPC4 was clarified by sequencing of the intron-exon transitions and is shown by way of example in Figure 2.
  • In situ hybridizations with a Fragments from the coding region of the OTRPC4-DNA 0 showed a high expression of the OTRPC4-RNA in the distal bundle of the kidney, but also plexus choreoid in the brain ventricles ( Figure 3).
  • GFP green fluorescent protein
  • pEGFP-NI fluorescent protein
  • the invention relates to a human OTRPC4 with the amino acid sequence: MADSSEGPRAGPGEVAELPGDESGTPGGEAFPLSSLANLFEGEDGSLSPSPADASRP
  • Expression plasmid containing OTRPC4 was transiently transfected and compared to non-transfected control cells 24-36 hours later.
  • Northern blots for the detection of OTRPC4 RNA in human tissues A commercial Northern blot from Clontech was hybridized with a probe mix based on a partial clone that had been isolated from a human salivary cDNA bank. The probe mix consisted of three fragments, the by restriction of the clone with Stu I. The fragments were 496 bp, 556 bp and 698 bp long and correspond to the three 3 'Stu I fragments of the human DNA of OTRPC4.
  • RNAs from twelve human tissues There were also filters (Clontech) with RNA from cardiovascular tissues, from tissues of the digestive system. and hybridized from tissues of the endocrine system. No clear signal was shown on these Northern blots.
  • HEK293 cells were transiently transfected with expression plasmid containing the OTRPC4-GFP fusion construct and then the concentration of the intracellular calcium concentration ([Ca 2+ ] i) using the FURA-2 Method measured using a monochromatic single cell calcium measuring station ( Figure 4).
  • the basal [Ca 2+ ] j in OTRPC4-expressing cells was significantly increased compared to the control cells (94 + 11 nM; 50 cells measured in three independent experiments versus 41 ⁇ 3 nM; 63 cells measured in three independent experiments).
  • OTRPC4 as a regulator of the osmolarity of the body's own liquids
  • OTRPC4 can regulate the osmolarity of the body's own fluids, which are formed by secretion, such as cerebrospinal fluid, the aqueous humor of the eye (and thus the intraocular pressure), the saliva of the salivary glands, etc.
  • secretion such as cerebrospinal fluid, the aqueous humor of the eye (and thus the intraocular pressure), the saliva of the salivary glands, etc.
  • hypoosmolarity is initiated with a calcium influx counter-regulating processes.
  • the present invention therefore also relates to the use of modulators of OTRPC4 or the biological activity of OTRPC4, in particular inhibitors or activators, for regulating the osmolarity of the body's own liquids, in particular cerebrospinal fluid, aqueous humor in the eye, and / or saliva.
  • the present invention further relates to pharmaceutical compositions which contain modulators, inhibitors and / or activators of OTRPC4, and the use of modulators, inhibitors and / or activators of OTRPC4 for the production of pharmaceutical compositions for prophylactic or therapeutic Regulation of the osmolarity of the body's own fluids, which are formed by secretion, in particular cerebrospinal fluid, aqueous humor in the eye, and / or saliva, for diseases or health conditions where this is necessary.
  • the OTRPC4-mediated change in the intracellular calcium concentration depends on the osmolarity of the extracellular solution
  • the current-voltage curve of the current activated by hypoosmolar solution had the same shape as that of the spontaneous current in cells expressing OTRPC4, but the reversal potential was shifted to a more positive potential (-5.6 + 0.7 mV).
  • Removal of sodium and calcium ions from the extracellular solution resulted in a complete but reversible block of the inward flow and reduced the outward flow components (see Figure 7).
  • the addition of hypotonic extracellular solution triggered a current flow that showed the properties of chloride channels that are activated by volume changes (27).
  • the activation of these strains could be completely inhibited by the addition of the chloride channel blocker NPPB (50 ⁇ M), while the cation streams triggered by hypotonic solution in cells expressing OTRPC4 were not influenced by this blocker.
  • hypotonic solution used to initiate the currents was replaced with hypotonic solutions containing either only sodium or only 20 mM calcium as cations.
  • the above-described eukaryotic expression plamide with the OTRPC4 cDNA additionally also contains a gene which confers resistance to the antibiotic G418 to the cells which are transfected with this plasmid.
  • HEK293 cells were transfected by lipofection as described above and stably expressing cells isolated by selection with G418. In order that the OTRPC4 channel is not constitutively active during the selection period, the HEK293 cells were cultivated in a medium whose osmolarity was set to 320 mosmol / 1.
  • the HEK293 cells stably expressing the OTRPC4 channel were sown in 384 well plates and the increase in the [Ca 2+ ] i triggered by the addition of hypotonic medium was measured in the cells with the aid of a fluorescence imaging plate reader (FLIPR).
  • FLIPR fluorescence imaging plate reader
  • Intron 11 located at base 1965, has a size> 5 kb, so that the DNA for the long arm can be obtained here.
  • Exon 12 with approx. 420 bp was tagged with accompanying LoxP sites for the purpose of conditional knock-out.
  • the OTRPC4 protein expressed in the conditional knock-out mice will be functionally inactive in vivo.
  • the anti-sense sequences listed in Table 1 were selected according to the following rules:
  • GC and AT base pairs are roughly evenly distributed.
  • One of the sequences covers the ATG in order to include, in addition to the induction of an RNAse H, which degrades the target RNA, the inhibition of the translation start as a mechanism.
  • Antisense-Oligo 1 / Base 6-21 (5 ' -UTR) CGT CTG CAC TGC TCA G
  • the selected antisense sequences can also be used as flanking sequences for the construction of ribozymes.
  • Protein sequence against which the antibody is directed corresponds to distal C-terminus 0 of the murine OTRPC4 protein.
  • the peptide sequence is as follows:
  • rabbits were immunized with 1 mg of KLH conjugate of the peptide shown above.
  • the control cells were native HEK293 cells.
  • the fractions from the cytosol and the cholate extract showed no
  • HEK cells examined for the presence of the OTRPC4 protein in the intact cells.
  • the primary antibody was the peptide antibody described above.
  • As a secondary AK used an ant-rabbit AK with a FITC conjugate ( Figure).
  • Cell membrane is integrated as a native protein and the antibody recognizes the OTRPC4 protein and can detect it in Western blot or in the living cell via immunofluorescence.
  • Thastrup O. et al. Thapsigargin, a tumor promoter, discharges intracellular Ca 2+ strores by specific inhibition of the endoplasmic reticulum Ca2 + -ATPase. Proc. Natl. Acad. Be. USA 87: 2466-2470 (1990).
  • Figure 1 Amino acid sequence of the predicted pore-forming structure of OTRPC4 and the tissue distribution of the expression of OTRPC4. s
  • (a) The amino acid sequence of the predicted fifth and sixth transmembrane domains and the neighboring cytosolic domain of OTRPC4 is shown. The transmembrane regions 5 and 6 and the putative pore-forming cytoplasmic domain are designated as such, and the conserved amino acids are deposited
  • Figure 2 Sequences of the cDNA coding for OTRPC4 of the mouse and organization is of the genomic clone of OTRPC4. The translation start, the stop codons, as well as the transitions between exons and introns and the length of the introns are indicated. The amino acid sequence is shown below the DNA sequence, the predicted transmembrane regions and the ankyrin binding site are indicated.
  • a sagittal section (a) and a horizontal section (f) of an entire mouse kidney two enlargements of the sagittal section of the kidney (b, c), a sagittal section (e), a coronary section (f), a horizontal section (g) of an entire mouse -Brain and one
  • Figure 4 Increase in the intracellular calcium concentration in HEK293 cells transfected with a plasmid that expresses the cDNA of OTRPC4.
  • the intracellular calcium concentration was measured using the FURA-2 technique in cells that express OTRPC4 and compared to cells that do not express this channel.
  • 3s cells were initially cultured in isotonic solution containing 100mM mannitol and 1mM CaCl2.
  • the upper horizontal bar indicates the change of the extracellular solution with which the cells were washed to a 200 mM solution. The change in osmolarity was achieved by omitting the mannitol. In the period indicated by the lower horizontal bar, the calcium
  • the traces shown represent the mean values of 17 cells (for the OTRPC4-expressing cells) and 21 cells (for the control cells) in the same experiment.
  • the small figure shows the corresponding measurement traces for individual OTRPC4-expressing cells in the same experiment.
  • Figure 5 Osmolarity-dependent change in intracellular calcium concentration in HEK293 cells that transiently express OTRPC4. The maximum is shown Fluorescence quotients of the calcium-loaded and unloaded FURA-2 dye depending on the osmolarity of the extracellular solution.
  • Figure 6 Decrease in ion flow in OTRPC4-expressing cells in a hyperosmolar extracellular solution.
  • the ion flow was recorded by voltage ramps from -100 to +100 mM in a standard extracellular solution (osmolarity 305 mosmol / 1; 1) and after adding a solution containing mannitol with an osmolarity of 320 mosmol / 1 (2), the small picture shows the time course of the effect triggered by the increase in the osmolarity of the extracellular solution.
  • Figure 7 Increase in ion flow carried by cations triggered by hypotonic extracellular solution in cells that express OTRPC4.
  • A The whole cell ion current of an OTRPC4 expressing cell was measured at -100 and +100 mV. At the time indicated on the horizontal bar, the standard extracellular solution was exchanged for a solution containing 100 mM NaCl and 100 mM mannitol (osmolarity 320 mosmol / 1), then for a solution without mannitol (215 mosmol / 1 ) and then again with a hypoosmolar solution in which sodium and calcium were replaced by NMDG. Finally, the cell was rinsed again with 320 milliosmolar solution.
  • B The ion current is plotted, which was triggered by a single voltage ramp in an OTRPC4-expressing cell at the points in time denoted by numbers in (A).
  • FIG. 8 Western blot of OTRPC4 in subcellular fractions.
  • a western blot with three different fractions from HEK293 cells which had been transfected with the OTRPC4 was hybridized with the antibody.
  • the control cells were native HEK293 cells.
  • the fractions from the cytosol and the cholate extract showed no signal for the OTRPC4 fragment, the cell membrane fraction in the transfected HEK293 cells showed a clear signal for the occurrence of OTRPC4 in the cell membrane.
  • Figure 9 Increase in intracellular calcium concentration in cells expressing OTRPC4.
  • the first substance was added after 1 min, the second after 2 min.
  • the osmolarity of the measurement buffer was 320 mosmol / 1.
  • Figure 10 Increase in ion flow carried by cations triggered by hypotonic extracellular solution in cells of the choroid plexus.
  • the extracellular standard solution was exchanged for a solution which had an osmolarity of 300 mosmol / 1, then for a solution with 230 mosmol / and then again for the starting solution (osmolarity 300 mosmol /1).
  • the cells were stimulated with 20 ⁇ M serotonin in order to check the vitality of the cells and the sensitivity of the method.
  • the ratio-specific to non-specific fluorescence is plotted in a FURA-2 measurement against time.

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Abstract

La présente invention concerne des acides nucléiques qui codent les canaux cationiques non sélectifs OTRPC4, ainsi que des polypeptides qui sont codés par lesdits acides nucléiques. La présente invention concerne également des hôtes ou des cellules hôtes qui expriment ledit polypeptide, ainsi qu'un procédé de détection d'inhibiteurs, d'activateurs et de modulateurs desdits canaux cationiques OTRPC4. Cette invention concerne également des inhibiteurs, des activateurs et des modulateurs desdits canaux cationiques OTRPC4, ainsi que des compositions pharmaceutiques contenant lesdits inhibiteurs, activateurs et modulateurs. En outre, cette invention concerne des mammifères non humains, qui contiennent OTRPC4 en tant que transgène, que gène inactivé (knock-out) ou que gène modifié (knock-in).
PCT/EP2001/002837 2000-03-17 2001-03-14 Nouveau canal cationique non selectif WO2001068698A2 (fr)

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WO2003087158A3 (fr) * 2002-04-16 2004-06-10 Bayer Healthcare Ag Regulation de canal potentiel recepteur transitoire humain

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