| PATENT NUMBER | This data is not available for free |
| PATENT GRANT DATE | April 2, 2002 |
| PATENT TITLE |
DNA encoding a human subunit 5-HT3-C of the 5-HT3 serotonin receptor |
| PATENT ABSTRACT | DNA encoding human 5-HT3-C has been cloned and characterized. The recombinant protein is capable of forming biologically active human 5-HT3-C protein. The cDNA has been expressed in recombinant host cells that produce active recombinant protein. In addition, the recombinant host cells are utilized to establish a method for identifying modulators of the receptor activity, and receptor modulators are identified |
| PATENT INVENTORS | This data is not available for free |
| PATENT ASSIGNEE | This data is not available for free |
| PATENT FILE DATE | September 1, 1999 |
| PATENT REFERENCES CITED |
Mikayama et al. Proc. Natl. Acad. Sci. USA vol. 90, pp. 10056-10060, 1993.* Voet et al. Biochemistry, John Wiley & Sons, Inc., pp. 126-128 and 228-234.* E Barnard E.. A. (1996). The transmitter-gated channels: a range of receptor types and structures. Trends Pharmacol. Sci. 17, 305-309. Belelli, D., Balcarek, J. M., Hope, A. G., Peters, J. A., Lambert, J. J., and Blackburn, T. P. (1995). Cloning and functionaBelelll expression of a human 5-hydroxytrypstamine type 3 As receptor subunit. Mol. Pharmacol. 48, 1054-62. Boess, F. G., Steward, L. J., Steele, J. A., Liu, D., Reid, J., Glencorse, T. A., and Martin, I. L. (1997). Analysis of the ligand binding site of the 5-HT3 receptor using site directed mutagenesis: importance of glutamate 106. Neuropharmacology 36, 637-647. Bufton, K. E., Steward, L. J., Barber, P. C., and Barnes, N. M. (1993). Distribution and characterization of the [3H]granisetron-labeled 5-HT3 receptor in the human forebrain. Neuropharmacology 32, 1325-31. Davies, P. A., Pistis, M., Hanna, M. C., Peters, J. A., Lambert, J. J., Hales, T. G., and Kirkness, E. F. (1999). The 5-HT3B subunit is a major determinant of serotonin-receptor function. Nature (London) 397, 359-363. Derkach, V., Surprenant, A., and North, R. A. (1989). 5-HT3 receptors are membrane ion channels. Nature (London) 339, 706-9. Fletcher, S., and Barnes, N. M. (1998). Desparately seeking subunits: are native 5-H receptors really homomeric complexes? Trends Pharmacol. Sci. 19, 212-215. Fletcher, S., and Barnes, N. M. (1997). Purification of 5-hydroxytryptamine3 receptors from porcine brain. Br. J. Pharmacol. 122, 655-662. Fletcher, S., Lindstrom, J. M., Mckernan, R. M., and Barnes, N. M. (1998). Evidence that porcine native 5-HT3 receptors do not contain nicotinic acetylcholine receptor subunits. Neuropharmacology 37, 397-399. Furutani, M., Trudeau, M. C., Hagiwara, N., Seki, A., Gong, Q., Zhou, Z., Imamura, S.-i., Nagashima, H., Kasanuki, H., Takao, A., Momma, K., January, C. T., Robertson, G. A., and Matsuoka, R. (1999). Novel mechanism associated with an inherited cardiac arrhythmia: Defective protein trafficking by the mutant HERG (G601S) potassium channel. Circulation 99, 2290-2294. Gralla, R. J. (1998). Antiemetic therapy. Semin. Oncol. 25, 577-583. Greenshaw, A. J., and Silverstone, P. H. (1997). The non-antiemetic uses of serotonin 5-HT3 receptor antagonists: clinical pharmacology and therapeutic applications. Drugs 53, 20-39. Gurley, D. A., and Lanthorn, T. H. (1998). Nicotinic agonists competitively antagonized serotonin at mouse 5-HT3 receptors expressed in Xenopus oocytes. Neurosci. Lett. 247, 107-110. Hugnot, J.-P., Salinas, M., Lesage, F., Guillemare, E., de Weille, J., Heurteaux, C., Mattei, M.-G., and Lazdunski, M. (1996). Kv8.1, a new neuronal potassium channel subunit with specific inhibitory properties towards Shab and Shaw channels. Embo J. 15, 3322-3331. Jan, L. Y., and Jan, Y. N. (1997). Voltage-gated and inwardly rectifying potassium channels. In J. Physiol. (Cambridge, U. K.), pp. 267-282. Lambert, J. J., Peters, J. A., and Hope, A. G. (1995). 5-HT3 receptors. In Ligand- Voltage-Gated Ion Channels, R. North, ed.: CRC, Boca Raton, Fla), pp. 177-211. Lummis, S. C. R., and Baker, J. (1997). Radioligand binding and photoaffinity labeling studies show a direct interaction of phenothiazines at 5-HT3 receptors. Neuropharmacology 36, 665-670. Lummis, S. C. R., and Martin, I. L. (1992). Solubilization, purification, and functional reconstitution of 5-hydroxytryptamine3 receptors from N1E-115 neuroblastoma cells. Mol. Pharmacol. 41, 18-23. Lummis, S. C. R., Sepulveda, M. I., Kilpatrick, G. J., and Baker, J. (1993). Characterization of [3H]meta-chlorophenylbiguanide binding to 5-HT3 receptors in N1E-115 neuroblastoma cells. Eur. J. Pharmacol. 243, 7-11. Maricq, A. V., Peterson, A. S., Brake, A. J., Myers, R. M., and Julius, D. (1991). Primary structure and functional expression of the 5HT3 receptor, a serotonin-gated ion channel. Science (Washington, D. C., 1883-) 254, 432-7. Mathur, R., Zhou, J., Babila, T., and Koren, G. (1999). Ile-177 and Ser-180 in the S1 segment are critically important in Kv1.1 channel function. J. Biol. Chem. 274, 11487-11493. Miller, K., Weisberg, E., Fletcher, P. W., and Teitler, M. (1992). Membrane-bound and solubilized brain 5HT3 receptors: improved radioligand binding assays using bovine area postrema or rat cortex and the radioligands 3H-GR65630, 3H-BRL43694, and 3H-LY278584. Synapse (N. Y.) 11, 58-66. Miyake, A., Mochizuki, S., Takemoto, Y., and Akuzawa, S. (1995). Molecular cloning of human 5-hydroxytryptamine3 receptor: heterogeneity in distribution and function among species. Mol. Pharmacol. 48, 407-16. Passani, M. B., and Corradetti, R. (1996). Therapeutic potentials of itasetron (AU 6215), a novel 5-HT3 receptor antagonist, in the treatment of central nervous system disorders. CNS Drug Rev. 2, 195-213. Peters, J. A., Malone, H. M., and Lambert, J. J. (1992). Recent advances in the electrophysiological characterization of 5-HT3 receptors. Trends Pharmacol. Sci. 13, 391-7. Salinas, M., de Weille, J., Guillemare, E., Lazdunski, M., and Hugnot, J.-P. (1997). Modes of regulation of Shab K+ channel activity by the Kv8.1 subunit. J. Biol. Chem. 272, 8774-8780. Salinas, M., Duprat, F., Heurteaux, C., Hugnot, J.-P., and Lazdunski, M. (1997). New modulatory a subunits for mammalian Shab K+ channels. J. Biol. Chem. 272, 24371-24379. Shalaby, F. Y., Levesque, P. C., Yang, W.-P., Little, W. A., Conder, M. L., Jenkins-West, T., and Blanar, M. A. (1997). Dominant-negative KvLQT1 mutations underlie the LQT1 form of long QT syndrome. Circulation 96, 1733-1736. Shuck, M. E., Piser, T. M., Bock, J. H., Slightom, J. L., Lee, K. S., and Bienkowski, M. J. (1997). Cloning and characterization of two K + inward rectifier (Kir) 1.1 potassium channel homologs from human kidney (Kirl.2 and Kirl.3). J. Biol. Chem. 272, 586-593. Steward, L. J., West, K. E., Kilpatrick, G. J., and Barnes, N. M. (1993). Labeling of 5-HT3 receptor recognition sites in the rat brain using the agonist radioligand ([3H]meta-chlorophenylbiguanide. Eur. J. Pharmacol. 243, 13-18. Stocker, J., Hellwig, M., and Kerschensteiner, D. (1999). Subunit assembly and domain analysis of electrically silent K+ channel a-subunits of the rat Kv9 subfamily. J. Neurochem. 72, 1725-1734. Sugita, S., Shen, K. Z., and North, R. A. (1992). 5-Hydroxytryptamine is a fast excitatory transmitter at 5-HT3 receptors in rat amygdala. Neuron 8, 199-203. Turton, S., Gillard, N. P., Stephenson, F. A., and McKernan, R. M. (1993). Antibodies against the 5-HT3-A receptor identify a 54 kDa protein affinity-purified from NCB20 cells. Mol. Neuropharmacol. 3, 167-71. Van Hooft, J. A., and Vijverberg, H. P. M. (1995). Phosphorylation controls conductance of 5-HT3 receptor ligand-gated ion channels. Recept. Channels 3, 7-12. Waeber, C., Shoeffter, P., Hoyer, D., and Palacios, J. (1989). 5-HT3 receptors in the human brain- autoradiographic visualisation using [3H] ICS 205-930. Neuroscience 31, 393. Yakel, J. L., Shao, X. M., and Jackson, M. B. (1991). Activation and desensitization of the 5-HT3 receptor in a rat glioma .times. mouse neuroblastoma hybrid cell. J. Physiol. (London) 436, 293-308. Zerr, P., Adelman, J. P., and Maylie, J. (1998). Episodic ataxia mutations in Kvl.1 alter potassium channel function by dominant negative effects or haploinsufficiency. J. Neurosci. 18, 2842-2848. Shuck, Mary E.; Piser, Timothy M.; Bock, Jeffery H.; Slightom, Jerry L.; Lee, Kai S.; and Bienkowski, Michael J., "Cloning and Characterization of Two K.sup.+ Inward Rectifier (K.sub.ir) 1.1 Potassium Channel Homologs from Human Kidney (K.sub.ir 1.2 and K.sub.ir 1.3)", The Journal of Biological Chemistry, 1997, 586-593, vol. 272, No. 1, The American Society for Biochemistry and Molecular Biology, Inc., U.S.A. Mathur, Rajesh; Zhou, Jun; Babila, Tamar; and Koren, Gideon, "lle-177 and Ser-180 in the S1 Segment are Critically Important in Kv1.1 Channel Function", The Journal of Biological Chemistry, 1999, 11487-11493, vol. 274, No. 17, The American Society for Biochemistry and Molecular Biology, Inc., U.S.A. Stocker, Martin; Hellwig, Michaela; and Kerschensteiner, Daniel, "Subunit Assembly and Domain Analysis of Electrically Silent K.sup.+ Channel (alpha)-Subunits of the Rat Kv9 Subfamily", Journal of Neurochemistry, 1999, 1725-1734, vol. 72(4), Lippincott, Williams & Wilkins. Zerr, Patricia; Adelman, John P.; and Maylie, James, "Episodic Ataxia Mutations in Kv1.1 Alter Potassium Channel Function by Dominent Negative Effects or Haploinsufficiency", The Journal of Neuroscience, 1998, 2842-2848, vol. 18(8), Society for Neuroscience. Shalaby, Fouad Y.; Levesque, Paul C.; Yang, Wen-Pin; Little, Wayne A.; Conder, Mary Lee; Jenkins-West, Tonya; and Blanar, Michael A., "Dominant-Negative KvLQT1 Mutations Underlie the LQT1 Form of Long QT Syndrome", Circulation, 1997, 1733-1736, vol. 96(6), Lippincott, Williams & Wilkins. Furutani, Michiko; Trudeau, Matthew C.; Hagiwara, Nobuhisa; Seki, Akiko; Gong, Qiuming; Zhou, Zhengfeng; Imamura, Shin-ichiro; Nagashima, Hirotaka; Kasanuki, Hiroshi; Takao, Atsuyoshi; Momma, Kazuo; January, Craig T.; Robertson, Gail A.; and Matsuoka, Rumiko, "Novel Mechanism Associated with an Inherited Cardiac Arrhythmia: Defective Protein Trafficking by the Mutant Herg (G601S) Potassium Channel", Circulation, 1999, 2290-2294, vol. 99(17), Lippincott, Williams & Wilkins. Salinas, Miguel; Duprat, Fabrice; Heurteaux, Catherine; Hugnot, Jean-Philippe; and Lazdunski, Michel, "New Modulatory .alpha. Subunits for Mammalian Shab K.sup.+ Channels", The Journal of Biological Chemistry, 1997, 24371-24379, vol. 272, No. 39, The American Society for Biochemistry and Molecular Biology, Inc., U.S.A. Hugnot, Jean-Philippe; Salinas, Miguel; Lesage, Florian; Guillemare, Eric; De Weille, Jan; Heurteaux, Catherine; Mattel, Marie-Genevieve; and Lazdunski, Michel, "Kv8.1, A New Neuronal Potassium Channel Subunit with Specific Inhibitory Properties Towards Shab and Shaw Channels", The EMBO Journal, 1996, 3322-3331, vol. 15, No. 13, Oxford University Press. Salinas, Miguel; De Weille, Jan; Guellemare, Eric; Lazdunski, Michel; and Hugnot, Jean-Philippe, "Modes of Regulation of Shab K.sub.+ Channel Activity by the Kv8.1 Subunit", The Journal of Biological Chemistry, 1997, 8774-8780, vol. 272, No. 13, The American Socity for Biochemistry and Molecular Biology, Inc., U.S.A. Jan, Lily Yeh and Jan, Yuh Nung, "Voltage-Gated and Inwardly Rectifying Potassium Channels", Journal of Physiology, 1997, 267-282, vol. 505, No. 2. Sugita, S.; Shen, K.-Z.; and North, R.A., "5-Hydroxytryptamine is a Fast Excitatory Transmitter at 5-HT.sub.3 Receptors in Rat Amygdala", Neuron, 1992, 199-203, vol. 8, Cell Press. Greenshaw, Andrew J and Silverstone, Peter H., "The Non-Antiemetic Uses of Serotonin 5-HT.sub.3 Receptor Antagonists", Drugs, 1997, 20-39, vol. 53(1), Adis International Limited. Bufton, Kate E.; Steward, Lucinda J.; Barber, Peter C.; and Barnes, Nicholas M., "Distribution and Characterization of the [.sup.3 H]Granisetron-Labelled 5-HT.sup.3 Receptor in the Human Forebrain", Neuropharmacology, 1993, 1325-1331, vol. 32, No. 12, Pergamon Press Ltd., Great Britain. |
| PATENT CLAIMS |
What is claimed is: 1. An isolated and purified DNA molecule that encodes human 5-HT3-C protein, wherein said protein comprises the amino acid sequence set forth in SEQ ID NO: 9. 2. The isolated and purified DNA molecule of claim 1, having a nucleotide sequence selected from a group consisting of SEQ ID NO: 4 and SEQ ID NO: 5. 3. An expression vector for expression of a human 5-HT3-C protein in a recombinant host, wherein said vector contains a recombinant nucleic acid molecule encoding human 5-HT3-C protein, wherein said protein comprises the amino acid sequence set forth in SEQ ID NO: 9. 4. The expression vector of claim 3, comprising a nucleotide sequence selected from a group consisting of SEQ ID NO: 4 and SEQ ID NO: 5. 5. A process for expression of human 5-HT3-C protein in a recombinant host cell, comprising: (a) transferring the expression vector of claim 3 into suitable host cells; and (b) culturing the host cells under conditions that allow expression of the human 5-HT3-C protein from the expression vector. 6. A recombinant host cell containing a recombinantly cloned nucleic acid molecule encoding human 5-HT3-C protein, wherein said protein comprises an amino acid sequence set forth in SEQ ID NO: 9. 7. The recombinant host cell of claim 6, wherein said recombinantly cloned nucleic acid molecule has a nucleotide sequence selected from a group consisting of SEQ ID NO: 4 and SEQ ID NO: 5. -------------------------------------------------------------------------------- |
| PATENT DESCRIPTION |
BACKGROUND OF THE INVENTION Serotonin (5-hydroxytryptamine, 5-HT) is a multifunctional chemical transmitter that signals though cell surface receptors. At least fourteen subtypes of serotonin receptors have been defined pharmacologically (Julius, 1996). Thirteen of the fourteen known receptors are G-protein coupled receptors and the only known ionotropic 5-HT receptor, the type 3 5-HT3 receptor, is a fast activating, ligand gated non-selective cation channel unique among known monoamine receptors (Derkach et al., 1989). The 5-HT3 receptor is exclusively localized on neurons in the central (Waeber et al., 1989; Yakel et al., 1991) and peripheral (Fozard, 1984) nervous systems. Activation of the 5-HT3 receptor leads to membrane depolarization and an increase in intracellular Ca.sup.2+. The 5-HT3 receptor is the target of antagonists (granisetron and ondansetron) selective against the nausea induced by cytotoxic chemotherapy and general anesthesia (Gralla, 1998). There is some evidence that serotonin 5-HT3 receptors are important in pain reception, anxiety, cognition, cranial motor neuron activity, sensory processing, modulation of affect, and the behavioral consequences of drug abuse (Greenshaw and Silverstone, 1997; Lambert et al., 1995; Passani and Corradetti, 1996). There are two known subunits for the human 5-HT3 receptor: 5-HT3-A (Belelli et al., 1995; Miyake et al., 1995) and 5-HT3-B (Davies et al., 1999). They have structural and functional similarities with nicotinic, GABA-ergic and other ligand gated ion channels (Barnard, 1996; Gurley and Lanthorn, 1998; Maricq et al., 1991). The modifier subunit 5-HT3-B for the serotonin 5-HT3 receptor can explain certain observations that 5-HT3 receptors from a variety of preparations have distinct pharmacological, kinetic, permeation and voltage-dependent properties (Peters et al., 1992) (Dubin et al., manuscript submitted to JBC). However, biochemical evidence suggests that 5-HT3 receptors may exist as heteromultimers composed of more than 2 subunits. Receptors purified from a variety of sources by affinity chromatography usually reveal at least 2 major protein bands with molecular masses in the order of 54 and 38 kDa (Lambert et al., 1995). The 5-HT3-A receptor corresponds to the former (Turton et al., 1993). Affinity purified 5-HT3 receptor solubilized from pig cerebral cortex is composed of at least 3 separable components, based on silver staining of proteins on denaturing gels (Fletcher and Barnes, 1997). A number of these protein bands are not recognized by specific antibodies directed against the recombinant 5-HT3-A subunit (Fletcher and Barnes, 1997), and their sizes are too large (52-71 kDa) to be considered as degraded 5-HT3-A fragments (Fletcher and Barnes, 1998). Heteromeric assembly of subunits often produces channels with altered function compared to homomeric channels. An increasing number of subunits have been reported to be electrically silent when expressed alone but profoundly inhibit the function of specific classes of channels in co-expression studies (Kv8, Kv9, Kir1.3) (Hugnot et al., 1996; Salinas et al., 1997; Salinas et al., 1997; Shuck et al., 1997; Stocker et al., 1999). Like 5-HT3 receptors, voltage-gated potassium channels are heteromers of similar subunits (Jan and Jan, 1997). Kv8.1 and members of the Kv9 family have the capability to abolish the functional expression of specific potassium channel family members when co-expressed at high levels (Hugnot et al., 1996; Salinas et al., 1997; Salinas et al., 1997; Stocker et al., 1999). Immunoprecipitation experiments reveal co-assembly of Kv8.1 with Kv2 (Hugnot et al., 1996). Kv8.1 does not appear to reach the plasma membrane when expressed alone (Salinas et al., 1997) and homomeric assembly of Kv9 subunits does not occur (Stocker et al., 1999). Functional expression of the inward potassium channel Kir1.3 in Xenopus oocytes was not detectable, however, co-expression of Kir1.3 with either Kir1.1 or Kir1.2 reduced the currents resulting from expression of these inward-rectifier subunits alone, consistent with a negative influence on Kir1.1 and Kir1.2 expression (Shuck et al., 1997). A number of naturally occurring mutant channels exist that reduce channel function. In one form of episodic ataxia, mutations in the gene encoding Kv1.1 abolish the function of wild-type Kv1.1 subunits (Mathur et al., 1999; Zerr et al., 1998). In the LQT1 form of long QT syndrome KvLQT1 mutations A177P or T311I have a dominant negative effect on currents produced by KvLQT1 with or without minK when channel subunit combinations are expressed in Xenopus oocytes (Shalaby et al., 1997). Expression of these KvLQT1 mutants either individually or in combination yielded inactive channels when expressed individually and inhibit wild-type KvLQT1 currents in a dominant-negative fashion. A naturally occurring HERG mutant G601S is a hypomorphic mutation (ie., a specific mutant form of a gene that exhibits function qualitatively similar to the normal state but with quantitatively less function), resulting in a reduced current amplitude and represents a novel mechanism underlying LQT2 (Furutani et al., 1999). A wide range of single channel conductances has been reported for the 5-HT3-A receptor subunit and endogenous 5-HT3 receptors in native cells. This variation is attributable in part to heteromeric formation of 5-HT3-A with 5-HT3-B subunits (Davies et al., 1999). Homomeric receptors revealed a sub-pS conductance whereas heteromeric receptors displayed large single channel conductance (16 pS) (Davies et al., 1999). Modulation of the conductance of 5-HT3 receptors has been reported. Van Hooft and colleagues have shown that the conductance of 5-HT3 receptors in N1E-115 cells is dependent on phosphorylation conditions (Van Hooft and Vijverberg, 1995). An unmet need in this field is the ability to reconstitute a recombinant expression system that fully mimics the 5-HT3 receptor complex in natural tissue, likely due to incomplete receptor complexes localized at the cell surface. By creating a more physiologically relevant model of the 5-HT3 receptor complex using in vitro systems, it will be easier to develop and test new therapeutic compounds for treatment of diseases associated with the 5-HT3 serotonin receptor. This unmet need has been met by the isolation and characterization of a novel 5-HT3 receptor subunit cDNA molecule, hereafter termed 5-HT3-C. Using a recombinant expression system, functional DNA molecules encoding the subunit have been isolated. The biological and structural properties of this protein are disclosed, as is the amino acid and nucleotide sequence. The recombinant protein is useful to identify modulators of the 5-HT3 serotonin receptor complex, and to reconstitute a more realistic physiological 5-HT3 receptor response in recombinant systems. Co-expression of the 5-HT3-A with the 5-HT3-C subunit reduces the biological function of the 5-HT3-A receptor for some known modulators of 5-HT3 receptors. Modulators identified in the assay disclosed herein are useful as therapeutic agents. The recombinant DNA molecules, and portions thereof, are useful for isolating homologues of the DNA molecules, identifying and isolating genomic equivalents of the DNA molecules, and identifying, detecting or isolating mutant forms of the DNA molecules. SUMMARY OF THE INVENTION A DNA molecule encoding a human subunit with homology to the 5-HT3-A serotonin receptor has been cloned and when co-expressed with the short form of the human serotonin 5-HT3-A receptor modifies the function of the 5-HT3 receptor. Using a recombinant expression system, functional DNA molecules encoding the human serotonin 5-HT3 receptor modifier protein (5-HT3-C) have been isolated. The biological and structural properties of these proteins are disclosed, as is the amino acid and nucleotide sequence. The recombinant protein is useful to identify modulators of human 5-HT3 receptors composed of both 5-HT3 receptors and the modifier subunit (5-HT3-C). Modulators identified in the assay disclosed herein are useful as therapeutic agents for conditions including, but are not limited to, nausea, depression, anxiety, psychoses (for example schizophrenia), urinary continence, Huntington's chorea, tardive dyskinesia, Parkinson's disease, obesity, hypertension, migraine, Gilles de la Tourette's syndrome, sexual dysfunction, drug addiction, drug abuse, cognitive disorders, learning, Alzheimer's disease, cerebral coma, senile dementia, obsessive-compulsive behavior, panic attacks, pain, social phobias, eating disorders and anorexia, cardiovascular and cerebrovascular disorders, non-insulin dependent diabetes mellitus, hyperglycemia, constipation, arrhythmia, disorders of the neuroendocrine system, stress, and spasticity, as well as acid secretion, ulcers, airway constriction, asthma, allergy, inflammation, and prostate dysfunction. The recombinant DNA molecules and portions thereof, are useful for isolating homologues of the DNA molecules, identifying and isolating genomic equivalents of the DNA molecules, gene therapy applications, and identifying, detecting or isolating mutant forms of the DNA molecules. |
| PATENT EXAMPLES | This data is not available for free |
| PATENT PHOTOCOPY | Available on request |
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