MXPA00006974A - Nucleic acids encoding a functional human purinoreceptor p2x3 - Google Patents

Abstract

A human P2X3 purinergic receptor polypeptide is provided. Nucleic acid molecules encoding the human P2X3 receptor polypeptide, and vectors and host cells containing such nucleic acid molecules, are also provided. In addition, methods are provided for producing the P2X3 receptor polypeptide, as are methods of using such polypeptides and host cells that express the same to screen for compounds having activity at the P2X3 receptor.

Description

NUCLEIC ACIDS CODING A FUNCTIONAL HUMAN P2X3 PURINORECEPTOR AND METHODS OF PRODUCTION AND USE OF SAME TECHNICAL FIELD The invention relates generally to receptor proteins and to DNA and RNA molecules that code for them. In particular, the invention relates to a nucleic acid encoding a P2X3 receptor. The invention also relates to methods for using the encoded P2X to thereby identify compounds that interact with it.

BACKGROUND OF THE INVENTION P2 receptors have been generally classified as either metabotropic nucleotide receptors or ionotropic receptors for extracellular nucleotides. It is believed that metabotropic nucleotide receptors (usually designated P2Y or P2Yn, where "n" is an integer subscript indicating the subtype) differ from ionotropic receptors (usually designated P2X or P2Xn) in that they are based on a fundamentally different means of transduction. of transmembrane signal: P2Y receptors operate through a G-protein coupled system, whereas P2X receptors are ligand-gate ion channels. The ligand for these P2X receptors is ATP, and / or other natural nucleotides, for example, ADP, UTP, UDP, or synthetic nucleotides, for example, 2-methylthioATP. At least seven P2X receptors and the cDNA sequences encoding them have been identified to date. The P X? of cDNA was cloned from the smooth muscle of the rat vas deferens (Valera et al. (1994) Nature 371: 516-519) and the cDNA P2X2 was cloned from PC12 cells (Brake et al. (1994) Nature 371: 519-523). Five other P2X receptors have been found in the rat cDNA literature by virtue of their sequence similarity to 2Xi and P2X2 (P2X3: Lßwis et al. (1995) Nature 377: 432-435, Chen et al. (1995) Nature 377: 428 -431; P2X4: Buell et al. (1996) EMBO J. 15: 55-62, Seguela et al. (1996) J. Neurosci 16: 448-455, Bo et al. (1995) FEBS Lett 375: 129-133 , Soto et al. (1996) Proc. Nati, Acad. Sci. USA 93: 3684-3688, Wang et al. (1996) Biochem. Biophys. Res. Commun. 220: 196-202; P2X5: Collo et al. (1996) J. Neurosci 16: 2495-2507, García-Guzmán et al. (1996) FEBS Lett 388: 123-127, P2X6 Collo et al. (1996), supra, Soto et al. (1996) Biochem. Biophys. Res. Commun. 223: 456-460; P2X7: Suprenant et al. (1996) Science 272: 735-738). For a comparison of the amino acid sequences of P2X receptors see Buell et al. (1996; Eur. J. Ne osci. 8: 2221-2228) Native P2X receptors rapidly form activated, non-selective cationic channels that are activated by ATT. Rat P2X? And rat P2X2 have equal permeability to Na + and K +, but significantly less for Cs + .The channels formed by the P2X receptors generally have high permeability for Ca + (Pca P to 4) .The P2X ?, P2X2 and Cloned rat P2X4 exhibit the same permeability for Ca2 + as observed with native receptors, however, the mechanism by which P2X receptors form an ionic pore or agglutinate ATP is not known.A variety of tissues and cell type, including epithelial, immune , muscular and neuronal, express at least one form of P2X receptor In the rat, the P2X3 receptor distribution seems to be mainly in sensory ganglia such as the dorsal, trigeminal root ganglia is and nodosos. However, the study of the role of individual P2X receptors is hindered by the lack of receptor-specific subtype agonists and antagonists. For example, a useful agonist for studying ATP gate channels is a, β-methylene-ATP (a.βmeATP). However, P2X receptors exhibit differential sensitivity for the P2X agonist? and 2X2 which are sensitive and insensitive to a.βmeATP, respectively. The predominant forms of P2X receptors in the rat brain, P2X4 and P2X6 receptors, they can not be blocked by suramin or PPADS. These two forms of the P2X receptor are also not activated by a.βmeATP and are, thus, inextricable for study with commonly available pharmacological tools. A therapeutic role has been suggested for P2 receptors, for example, for cystic fibrosis (Boucher et al. (1995) in: Belardinelli and collaborators (eds) Adenosine and Adenine Nucleotides: from Molecular Biology you tntegrative Physiology (Kluwer Acad., Norwetl MA) pp 525-532) diabetes (Loubatiéres-Mariani et al. (1995) in: Belardinelli et al. (eds), supra, pp. 337-345), immune and inflammatory diseases (Di Virgilio et al. (1995) in: Bellardinefli et al. eds), supra, pp 329-335), cancer (Rapaport (1993) Drug Dev.Res.28: 428-431), constipation and diarrhea (Milner et al. (1994) in: Kamm et al. (eds), Constipation and Related Disorders: Pathophysiofogy and Management in Adults and Children (Wrightson Biomedical, Bristol) pp 41-49), behavioral disorders such as epilepsy, depression and degenerative diseases associated with aging (Williams (1993) Drug Dev. Res. 28: 438- 444), contraception and sterility (Foresta and collaborators (1992) J. Biol. Chem. 257: 19443-19447), and wound healing (Wang et al. (1990) Biochim. Biophys. Res. Commun. 166: 251-258). Accordingly, there is a need in the art for specific agonists and antagonists for each P2 receptor subtype and, in particular, agents that will be effective in vivo, as well as for methods for identifying specific agonist and antagonist compounds for P2 receptor.

BRIEF DESCRIPTION OF THE INVENTION The present invention relates to a human P2X3 receptor. In one embodiment, a DNA molecule or fragments thereof is provided, wherein the DNA molecule encodes a human P2X3 receptor or a subunit thereof. In another embodiment, a recombinant vector comprising such a DNA molecule, or fragments thereof, is provided. In another embodiment, the subject invention is directed to a human P2X3 receptor polypeptide, either alone or in multimeric form.

In still other embodiments, the invention is directed to messenger RNA encoded by the DNA, recombinant host cells transformed or transfected with vectors comprising the DNA or fragments thereof, and methods for producing P2X3 polypeptides using such cells. In yet another embodiment, the invention is directed to a method for expressing a human P2X3 receptor, or a subunit thereof, in a cell to produce the resulting P2X3-containing receptor. In a further embodiment, the invention is directed to a method for using such cells to identify potentially therapeutic compounds that modulate or otherwise interact with the above P2X3-containing receptors. These and other embodiments of the present invention will readily occur to those of ordinary skill in the art in view of the description herein.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 represents the sequence of the product P2X3 5'RACE of Example 2 (SEQ ID NO: 13), in which the primary sequences are underlined and the predicted initiation codon (ATG) is shown in bold . Figure 2 represents the sequence of the product P2X3 3'RACE of Example 3 (SEQ ID NO: 14), in which the primary sequences are underlined and the predicted stop codon (TAG) is shown in bold.

Figure 3 depicts the sequence of the complete open reading frame of CDNA encoding the human P2X3 receptor polypeptide (SEQ ID NO: 15). The initiation (ATG) and termination (TAG) codons are shown in bold type: the 5 'and 3' flanking sequences introduced during the construction of the plasmid, including the restriction sites EcoRI (GAATTC) and Not I (GCGGCCGC), are underlined. Figure 4 depicts the predicted aligned amino acid sequences of human P2X3 (hP2X3) (SEQ ID NO: 16) and (rP2X3) (SEQ ID NO: 17) polypeptide receptors of rat. Identical waste is identified by use of cash.

DETAILED DESCRIPTION OF THE INVENTION The practice of the present invention will employ, unless otherwise specified, conventional techniques of molecular biology, microbiology, recombinant DNA technology, electrophysiology, and pharmacology, which are within the skill of the art. Such techniques are fully explained in the literature. See, for example, Sambrook, Fr? Tsch & Mahiatis, Molecular Cloning: A Laboratory Manual, second edition (1989); DNA Ctoning, Vols. I and ll (D.N.GIover ed., 1985); Perbal, B., A Practical Guide to Molecular Cloning (1984); Ta series, Methods In Enzymology (S. Colowick and N. Kaplan eds., Academic Press, Inc.); Transcription and Translation (Hames et al., Eds. (1984); Gene Transfer Vectors for Mammalian Cells (JH Miller et al., Eds. (1987) Cold Spring Harbor Laboratory, Cold Spring Harbor, NY); Scopes, Protein Purifications: Principáis and Practice (2aed., Springer-Verlag) and PCR: JA Practical Approach (McPherson et al., Eds. (1991) IRL Press.) All patents, applications and patent publications cited herein, whether supra or infra, are incorporated herein by reference in their entirety and are considered representative of the prevailing state of the art As used in this specification and the appended claims, the singular forms "a," "an," and "the," include plural references unless the content clearly dictates otherwise, for example, the reference to "a primary" includes two or more such primary, the reference to "an amino acid" includes more than one of such an amino acid, and the like. In describing the present invention, the following terms will be employed, and are intended to be defined as indicated below. The term "P2 receptor" is intended for a purinergic receptor of the ATP ligand and / or other purine or pyrimidine nucleotides, either natural or synthetic. P2 receptors are broadly subclassified as * P2X "or" P2Y "receptors.These types differ in their pharmacology, structure, and signal transduction mechanisms.P2X receptors are generally gate-ligand ion channels, whereas the receptors P2Y generally operate through a G-coupled protein system Furthermore, and without pretending to be limited by any theory, it is believed that P2X receptors comprise multimers of polypeptide receptors, said multimers may be of the same or different subtypes. Accordingly, the term "P2X receptor" refers, appropriately, to the individual subunit or receptor subunits, as well as to the heteromeric and homomeric receptors encompassed thereby The term "P2Xn" means a subtype of P2X receptor wherein n is a of at least 1. At the time of the invention, at least 7 P2X receptor subtypes have been isolated and / or characterized. "A" rectal agonist. eptor P2X3"is a compound that agglutinates to and activates a P2X3 receptor. By "active" is meant the provocation of one or more pharmacological, physiological, or electrophysiological responses. Such responses may include, but are not limited to, an increase in receptor-specific cellular depolarization. A "P2X3 receptor antagonist" is a substance that binds to a P2X3 receptor and prevents agonists from activating the receptor. Pure antagonists do not activate the receptor, but some substances may have mixed agonist and antagonist properties. The term "polynucleotide" as used herein means a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. This term refers only to the primary structure of the molecule. Thus, the term includes double and single filament DNA, as well as double and single filament RNA. It also includes modifications, such as by methylation and / or by coping, and unmodified forms of the polynucleotide. The term "variant" is used to refer to an oligonucleotide sequence that differs from the wild type sequence related to the insertion, deletion or substitution of one or more nucleotides. When not caused by a structurally conservative mutation (see below), such a variant oligonucleotide is expressed as a "protein variant" which, as used herein, denotes a polypeptide sequence that differs from the wild-type polypeptide in the insertion, elimination or substitution of one or more amino acids. The protein variant differs in primary structure (amino acid sequence), but may or may not differ significantly in secondary or tertiary structure or function in relation to the wild type. The term "mutant" generally refers to an organism or cell that exhibits a new genetic character or phenotype as the result of a change in its gene or chromosome. In some cases, however, "mutant" can be used with reference to a variant protein or oligonucleotide and "mutation" can refer to the change underlying the variant. "Pipeptide" and "protein" are used interchangeably herein and indicate a molecular chain of amino acids linked through peptide ligations. The terms do not refer to a specific length of polypeptide. The terms include post-translational modifications of the polypeptide, for example, glycosylations, acetylations, phosphorylations and the like. In addition, protein fragments, analogs, mutated proteins or variants, fusion proteins and the like are included within the meaning of polypeptide, provided that such fragments, etc., retain the agglutination or other characteristics necessary for their intended use.

A "functionally conservative mutation" as used herein means a change in a polynucleotide encoding a derivatized polypeptide in which the activity is not ubstantially altered as compared to that of the polypeptide from which the derivative is made. Such derivatives can have, for example, insertions, deletions, or amino acid substitutions in the relevant molecule that does not substantially affect its properties. For example, the derivative may include conservative amino acid substitutions, such as substitutions that retain the general charge, hydrophobicity / hydrophilicity, side chain portion, and / or steric volume of the substituted amino acid, eg, Gli / Ala, Val / lle / Leu, Asp / Glu, Lis / Arg, Asn / Gln, Tr / Ser, and Fe / Trp / Tir. By the term "structurally conservative mutant" is meant a polynucleotide that contains changes in the sequence of nucleic acids, but which encode a polypeptide having the same amino acid sequence as the polypeptide encoded by the polynucleotide from which the variant is derived degenerate. This can occur because a specific amino acid can be encoded by more than one "codon", or sequence of three nucleotides, that is, due to the degeneracy of the genetic code. "Recombinant host cells", "host cells", "cells", "cell lines", "cell cultures", and other such terms indicating microorganisms or higher eukaryotic cell lines grown as unicellular entities refer to cells that they can be, or have been, used as receptacles for recombinant vectors or other transfer DNA, independently of the method by which the DNA is introduced into the cell or the subsequent arrangement of the cell. The terms include the progeny of the original cell which has been transfected. Cells in primary culture as well as cells such as oocytes can also be used as containers. A "vector" is a repetition in which another segment of polynucleotide, tat, is bound to bring about duplication and / or expression of the joined segment. The term includes expression vectors, cloning vectors, and the like. A "coding sequence" is a polynucleotide sequence that is transcribed into RNA and / or translated into a polypeptide. The boundaries of the coding sequence are determined by a translation initiation codon at the 5'- terminus and a translation high codon at the 3'- terminus. A coding sequence can include, but is not limited to, mRNA, cDNA , and recombinant polynucleotide sequences. Variants or analogs can be prepared by removing a portion of the coding sequence, by inserting a sequence, and / or by substituting one or more nucleotides within the sequence. Techniques for modifying nucleotide sequences, such as site-directed mutagenesis, are well known to those skilled in the art. See, for example, Sambrook et al., Supra: DNA Cloning, vols. I and II, supra: Nucleic Acid Hybridization, supra. "Operably linked" refers to a situation where the described components are in a relationship that allows them to function in their intended manner. Thus, for example, a control sequence "operably linked" to a coding sequence is linked in such a way that the expression of the coding sequence is achieved under conditions compatible with the control sequences. A coding sequence can be operably linked to control sequences that direct the transcription of the polynucleotide whereby said polynucleotide is expressed in a host cell. The term "transfection" refers to the insertion of an exogenous polynucleotide into a host cell, regardless of the method used for the insertion, or the molecular form of the polynucleotide that is inserted. Included are the insertion of a polynucleotide per se and the insertion of a plasmid or vector comprised of the exogenous polynucleotide. The exogenous polynucleotide can be transcribed and translated directly by the cell, maintained as a non-integrated vector, for example, a plasmid, or alternatively, can be stably integrated into the host genome. "Transfection" is generally used with reference to a eukaryotic cell while the term "transformation" is used to refer to the insertion of a polynucleotide into a prokaryotic cell. "Transformation" of a eukaryotic cell can also refer to the formation of a cancerous or tumorigenesis state. The term "isolated", when referring to a polynucleotide or a polypeptide, is intended to mean that the indicated molecule is present in the substantial absence of other similar biological macromolecules. The term "isolated" as used herein means at least 75% by weight, more preferably at least 85% by weight, more preferably at least 95% by weight, and most preferably at least 98% by weight. % by weight of a composition is the isolated polynucleotide or polypeptide. An "isolated polynucleotide" that encodes a particular polypeptide refers to a polynucleotide that is substantially free of other nucleic acid molecules that do not encode the subject polypeptide; however, the molecule can include functionally and / or structurally conservative mutations as defined herein. A "test sample" as used herein means a component of a body of an individual that is a source of a P2X3 receptor. These test samples include biological samples that can be evaluated by the methods of the present invention described herein and include body fluids such as global blood, tissue and cell preparations. The following abbreviations of single-letter amino acids are used throughout the text: Alanine A Arginine R Asparigin N Aspartic Acid D Cysteine C Glutamine Q Glutamic Acid E Glycine G Histirin H Isoleusin I Leusin L Lysine K Meteonin M Phenylalanine F Proline P Serine S Threonine T Tryptophan W Tyrosine And Valine V A human P2X3 receptor, a polynucleotide encoding the variant receptor or polypeptide subunits thereof, and methods for making the receptor are provided herein. The invention includes not only the P2X3 receptor but also methods for filtering compounds using the receptor and cells expressing the receptor. In addition, polynucleotides and antibodies that can be used in methods for receptor detection, as well as reagents useful in these methods, are provided. Compounds and polynucleotides useful for regulating the receptor and its expression are also provided, as described hereinafter. In a preferred embodiment, the polynucleotide encodes a human P2X3 receptor peptide or a protein variant thereof that contains conservative amino acid substitutions. The DNA encoding the human P2X3 receptor and variants thereof can be derived from the genomic or cDNA, prepared by synthesis, or by a combination of techniques. The DNA can then be used to express the human P2X3 receptor or as a template for RNA preparation using methods well known in the art (see, Sambrook et al., Supra) or as a molecular probe capable of selectively hybridizing to, and thus detect the presence of, other nucleotide sequences encoding P2X3. The cDNA encoding the P2X3 receptor can be obtained from an appropriate DNA literature. The cDNA literatures can be investigated using the procedure described by Grunstein et al., (1975) Proc.Natt.Acad.Sci. E.U.A. 73: 3961. The thus obtained cDNA can then be modified and amplified using the pho- merase chain reaction ("PCR") and primary sequences to obtain the DNA encoding the human P2X3 receptor. More particularly, the PCR employs primary short oligonucleotides (generally 10 to 20 nucleotides in length) the matched opposite ends of a desired sequence within the DNA molecule. It is not necessary to know the sequence between the primaries. The initial annealing can be either RNA or DNA. If RNA is used, it is first inverted transcribed to cDNA. The cDNA is then denatured, using well-known techniques such as heat, and appropriate primary oligonucleotides are added in molar excess. The extension of primer is effected using DNA polymerase in the presence of deoxynucleotide triphosphates or analogous nucleotides. The resulting product includes the respective primaries in their 5 'terms, covalently linked to the synthesized complements of the original filaments. The duplicated molecule is denatured again, hybridized with primaries, and so on consecutively, until the product is sufficiently amplified. Such PCR methods are described in, for example, the US patents. Nos. 4,965,188; 4,800,159; 4,683,202; 4,683,195; incorporated herein by reference in their totalities. The PCR product is cloned and the clones containing the P2X3 receptor DNA, derived by segregation of the extended primary filament, are selected. The selection can be made using a primer as a hybridization probe. Alternatively, the P2X3 receptor DNA could be generated using an RT-PCR (reverse transcriptase polymerase chain reaction) approach starting with human RNA. Human RNA can be obtained from cells or tissue in which the P2X3 receptor is expressed, such as dorsal root ganglia, trigeminal ganglia, pituitary gland, ganglion nodosum or heart, using conventional methods. For example, the single stranded cDNA is synthesized from human RNA as the annealed using standard reverse transcriptase methods and the cDNA is amplified using PCR. This is but one example of the generation of P2X3 receptor variant from an RNA tuning of human tissue. Synthetic oligonucleotides can be prepared using an automated oligonucleotide synthesizer such as that described by Warner (1984) DNA 3: 401. If desired, Synthetic filaments can be labeled with P32 by treatment with polynucleotide kinase in the presence of P32-ATP, using standard conditions for the reaction. DNA Sequences, including those isolated from genomic or cDNA literature, can be modified by known methods which include site-directed mutagenesis as described by Zotler (1982) Nucleic Acids Res., 10: 6487. Briefly, the DNA to be modified is packaged in phage as a single strand sequence, and converted to a double strand DNA with DNA polymerase using, as a primer, a synthetic oligonucleotide complementary to the portion of the DNA to be modified, and which has the desired modification included in its own sequence. The culture of the transformed bacteria, which contains duplications of each filament of the phage, is coated with agar to obtain plaques. Theoretically, 50% of the new plates contain phage that has the mutated sequence, and the remaining 50% has the original sequence. The duplicates of the plates are hybridized for labeled synthetic probes at temperatures and conditions suitable for hybridization with the correct filament, but not with the unmodified sequence. The sequences that have been identified by hybridization are recovered and cloned. Alternatively, it may be necessary to identify clones by sequence analysis if there is difficulty in distinguishing the wild type variant by hybridization. In any case, the DNA would be of confirmed sequence. Once produced, the DNA encoding the P2X3 receptor can then be incorporated into a cloning vector or an expression vector for duplication in a suitable host cell. The construction of vectors employs methods known in the art. Generally, the site-specific DNA cleavage is performed by treating with suitable restriction enzymes under conditions that are generally specified by the manufacturer of these commercially available enzymes. After incubation with the restriction enzyme, protein is removed by extraction and the DNA recovered by precipitation. The split fragments can be separated using, for example, electrophoresis methods in polyacrylamide or agarose gel, according to methods known to those of skill in the art. Sticky terminal slit fragments can be smoothed at their ends using polymerase 1 of E. coli (Klenow) DNA in the presence of the appropriate deoxynucleotide triphosphates (dNTPs) present in the mixture. S1 nuclease treatment, resulting in the hydrolysis of any single filament DNA portions can also be used. Ligations are made using standard buffer and temperature conditions using T4 DNA and ATP tigase. Alternatively, restriction enzyme digestion of unwanted fragments can be used to prevent ligation. Standard vector constructs generally include elements of specific antibiotic resistance. The ligation mixtures are transformed into a suitable host, and the successful transformants selected by antibiotic or other resistance markers. Plasmids can then be prepared from the transformants according to methods known to those in the art which usually follow an amplification of chloramphenicol as reported by Clewell et al., (1972) J. Bacteriol. 110: 667. The DNA is usually isolated and analyzed by restriction enzyme analysis and / or sequencing. Sequencing can be done by the well-known dideoxy method of Sanger et al. (1977) Proc. Nati Acad. Sci. E.U.A. 74: 5463 as described further by Messing et al. (1981) Nucleic Acid Res. 9: 309, or by the method reported by Maxam et al. (1980) Meth.Enzymal. 65: 499. Problems with band compression, which are sometimes observed in regions rich in GC, are overcome by the use of, for example T-deazoguanosine or inosine, according to the method reported by Barr et al. (1986) Biotechniques 4: 428. The host cells are generally manipulated with the vectors of this invention, which can be a cloning vector or an expression vector. The vector may be in the form of a pfasmid, a viral particle, a phage, etc. The engineered host cells can be cultured in modified conventional nutrient medium as appropriate to activate promoters, select transformants / transfectants or amplify the subunit encoding polynucleotide. The culture conditions, such as temperature, pH and the like, are generally similar to those previously used with the host cell selected for expression, and will be apparent to those skilled in the art. Both prokaryotic and eukaryotic host cells can be used for expression of desired coding sequences when appropriate control sequences are used that are compatible with the designated host. For example, expression control sequences for prokaryotes include, but are not limited to, promoters, optionally containing operator portions, and ribosome binding sites. Transfer vectors compatible with prokaryotic hosts can be derived from, for example, the plasmid pBR322 which contains operons conferring resistance to ampicillin and tetracycline, and the various pUC vectors, which also contain sequences conferring markers with antibiotic resistance. These markers can be used to obtain successful transformants by selection. Commonly used prokaryotic control sequences include, but are not limited to, the lactose operon system (Chang et al. (1977) Nature 198: 1056), the tryptophan operon system (reported by Goeddel et al. (1980) Nucleic Acid Res. 8: 4057) and the promoter P1 derived from lamda and ribosome binding site of N gene (Shimatake and cotaboradors (1981) Nature 292: 128), the hybrid Tac promoter (De Boer et al. (1983) Proc. Nati. Acad. US Sc 292: 128) derived from sequences of the trp and tac UV5 promoters. The preceding systems are particularly compatible with E. coli; however, other prokaryotic hosts such as Basillus or Pseudomonas strains may be used if desired. Eukaryotic hosts include yeast and mammalian cells in culture systems. Yeast hosts Pichia pastoris, Saccharomyces cerevisiae and S. carlsbergensis are commonly used. Vectors compatible with yeast carry markers that allow the selection of successful transformants by conferring protopy to auxotropic mutants or resistance to heavy metals in wild-type strains. Vectors compatible with yeast can employ the 2-μ duplication origin (Broach and boilers (1983) Meth. Enzy oL 101: 307), the combination of CEN3 and ARSI or other means to ensure duplication, such as sequences that will result in incorporation of an appropriate fragment into the genome of the host cell. Control sequences for yeast vectors are known in the art and include, but are not limited to, promoters for the synthesis of glycolytic enzymes, including the promoter for 3-phosphoglycerate kinase. See, for example, Hess et al. (1968) J. Adv. Enzyme Reg. 7: 149, Holtand et al. (1978) Biochemistry 17: 4900 and Hitzeman (1980) J. Biol. Chem. 255: 2073. For example, some useful control systems are those that comprise the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) promoter or the dehydrogenase alcohol regulator (ADH), or the ADH2 / GAPDH hybrid yeast promoter described in Cousens et al. Gene (1987) 61: 265-275, terminators also derived from GAPDH, and, if secretion is desired, leader sequences of yeast alpha factor. In addition, the transcriptional regulatory region and the transcriptional initiation region that are operably linked can be such that they are not naturally associated in the wild-type organism. Mammalian cell lines are known in the art as hosts for expression and are available from depositories such as the American Type Culture Collection. These include, but are not limited to, HeLa cells, human embryonic kidney (HEK) cells, Chinese hamster ovary (CHO) cells, hamster pup kidney (BHK) cells, and others. Suitable promoters for mammalian cells are also known in the art and include viral promoters such as those of Simian Virus 40 (SV40), Rous sarcoma virus (RSV), adenovirus (ADV), bovine papilloma virus (BPV) and cytomegalovirus ( CMV). Mammalian cells may also require terminator sequences and addition poly A sequences; Increasing sequences that increase expression may also be included, and sequences that cause amplification of the gene may be desirable. These sequences are known in the art. Vectors suitable for duplication in mammalian cells may include viral replicates, or sequences that ensure integration of the appropriate sequences encoding the P2X3 receptor in the host genome. An example of such a mammalian expression system is described by Gopalakrishnan et al. (1995), Eur. J. Pharmacol. -Mol. Pharmacol. 290: 237-246. Other eukaryotic systems are also known, as are methods for introducing polynucleotides into such systems, such as amphibian cells, using standard methods such as described in Briggs et al. (1995) Neuropharmacol. 34: 583-590 or Stühmer (1992) Meth. Enzymol, 207: 319-345, insect cells using the methods described in Summers and Smith, Texas Agricultural Experiment Station Bulletin No. 1555 (1987), and the like. The baculovirus expression system can be used to generate high levels of recombinant proteins in insect host cells. This system allows the high level of protein expression, while the protein is post-translationally processed in a manner similar to mammalian cells. These expression systems use viral promoters that are activated following infection with baculovirus to boost the expression of cloned genes in insect cells (O'Reilly et al. (1992) Baculovirus Expression Vectors: A Laboratory Manual, IRL7 Oxford University Press). Transfection can be by any known method for introducing polynucleotides into a host cell, including packaging the polynucleotide in a virus and translating a host cell with the virus, by direct taking of the polynucleotide by the host cell, and the like, such methods are known to those skilled in the art. The selected transfection procedures depend on the host to be transfected and are determined by the routiner. The expression of the receptor can be detected by the use of a selective radioligand for the receptor. However, any radioligand binding technique known in the art can be used to detect the receptor (see, for example, Winzor et al. (1995) Quantitative Characterization of Lt.gand Biling, Wiley-Liss, Inc. NY; Michel et al. (1997) Mol. Pharmacol. 51: 524-532). Alternatively, expression can be detected using antibodies or functional measures, i.e., cell depolarization stimulated with ATP using methods that are well known to those skilled in the art. For example, influx of agonist-stimulated Ca2 +, or inhibition by agonist-stimulated Ca2 + influx antagonists, can be measured in mammalian cells transfected with the recombinant P2X3 receptor cDNA, such as COS, CHO or HEK cells. Alternatively, the influx of Ca + can be measured in cells that do not naturally express P2 receptors, for example, the human astrocytoma 1321 N1 cell line, but have been prepared using recombinant technology to transiently or stably express the P2X3 receptor. The P2X3 polypeptide is recovered and purified from cultures of recombinant host cells expressing thereto by known methods including ammonium sulfate or ethanol precipitation, acid extraction, anion exchange or cation chromatography, phosphochlo- those chromatography, hydrophobic interaction, hydroxyapatite chromatography or lectin chromatography. Protein redoubling steps may be used, as necessary, to complete the configuration of the protein. Finally, high performance liquid chromatography (HPLC) can be used for final purification steps. The human P2X3 receptor polypeptide, or fragments thereof, of the present invention can also be synthesized by conventional techniques known in the art, for example by chemical synthesis such as solid phase peptide synthesis. In general, these methods employ phase synthesis methods either solid or in solution. See, for example, J.M. Stewart and J.D. Young, So // cf Phase Peptide Synthesis, 2nd Ed., Pierce Chemical Co., Rockford IL (1984) and G. Barany and R.B. Merrifield, The Peptides; Analysis, Synthesis, Biology, editors E. Gross and J. Meienhofer, Vol. 2 Academic Press, New York, (1980), pp. 3-254, for solid phase peptide synthesis techniques; and M. Bodansky, Principies of Peptide Synthesis, Springer-Verlag, Berlin (1984) and E. Gross and J. Meienhofer, Eds. The Peptides, Analysis Synthesis, Biology, supra, Vol. 1 for synthesis in classical solutions. In a preferred system, either DNA or RNA derived therefrom, each encoding the human P2X3 receptor, can be expressed by direct injection into a cell, such as a Xenopus laevis oocyte. Using this method, the functionality of the human P2X3 receptor encoded by the DNA or mRNA can be evaluated as follows. A polynucleotide encoding the receptor is injected into an oocyte for translation to a functional receptor subunit. The function of the expressed variant human P2X3 receptor can be calculated in the oocyte by a variety of techniques including electrophysiological techniques such as voltage anchorage, and the like. The receptors expressed in a recombinant host cell can be used to identify compounds that modulate the activity of P2X3. In this regard, the specificity of the binding of a compound showing affinity for the receptor is demonstrated by measuring the affinity of the compound for cells expressing the receptor or membranes of those cells. This can be done by measuring the specific adhesion of the labeled (eg, radioactive) compound to the cells, cell membranes or isolated receptor, or by measuring the ability of the compound to displace the specific adhesion of a standard labeled ligand. See, Michel et al., Supra. Expression of variant receptors and filtration for compounds that bind to them, or inhibit the adhesion of labeled ligand to these cells or membranes, provide a method for rapid selection of compounds with high affinity for the receptor. These compounds can be agonists, receptor antagonists or modulators. Also, expressed receptors can be used to separate compounds that modulate P2X3 receptor activity. A method for identifying compounds that modulate P2X3 activity, comprises providing a cell expressing a human P2X3 receptor polypeptide, combining a test compound with the cell and measuring the effect of the test compound on the activity of the P2X3 receptor. The cell can be a bacterial cell, a mammalian cell, a yeast cell, an amphibian cell, an insect cell or any other cell that expresses the receptor. Preferably, the cell is a mammalian cell or an amphibian cell. Thus, for example, a test compound is evaluated for its ability to elicit an appropriate response, for example, the stimulation of cellular depolarization, or for its ability to modulate the response to an agonist or antagonist. Additionally, compounds capable of modulating P2X3 receptors are considered potential therapeutic agents in various disorders including, without limitation, conditions of the central nervous system or peripheral nervous system, for example, epilepsy, pain, depression, neurodegenerative diseases, and the like, and in disorders. of the reproductive system, asthma, peripheral vascular disease, hypertension, disorders of the immune system, irritable bowel disorder or premature ejaculation. In particular, P2X3 receptors have been implicated in the mediation of physiological pain responses (see Kennedy and Leff (1995) Nature 477: 385-386; Chen et al. (1995), supra). Accordingly, it is believed that the separation methods of the present invention may be especially suitable for the identification of compounds useful as analgesic and pain relieving agents. In addition, DNA, or RNA derived therefrom, can be used to design oligonucleotide probes for DNAs that express P2X3 receptors. As used herein, the term "probe" refers to a structure consisting of a polynucleotide, as defined above, which contains a nucleic acid sequence complementary to a nucleic acid sequence present in an objective polynucleotide. The polynucleotide regions of probes may be composed of DNA, and / or RNA, and / or synthetic nucleotide analogs. Such probes could be useful in in vitro hybridization assays to distinguish P2X3 variant of the wild-type message, provided that it can be difficult to design a method capable of making such a distinction given the small differences that may exist between sequences encoding the type wild and a P2X3 receptor variant. Alternatively, a PCR-based assay could be used to amplify the sample RNA or DNA for sequence analysis. In addition, the P2X3 polypeptide or fragment (s) thereof can be synthesized using conventional polypeptide synthesis techniques as is known in the art. Monoclonal antibodies that exhibit specificity and selectivity for the P2X3 polypeptide can be labeled with a measurable and detectable portion, eg, a fluorescent moiety, radiolabels, enzymes, chemiluminescent labels and the like, and used in in vitro assays. There are theories that such antibodies could be used to identify wild-type receptor or variant P2X3 polypeptides for immuno-diagnosis purposes. For example, antibodies have been generated to detect amyloid b1-40 v.1-42 in brain tissue (Wisniewskí et al. (1996) Biochem. J. 313: 575-580; see also Suzuki et al., (1994) Science 264: 1336-1340, Gravina et al. (1995) J. Biol. Chem. 270: 7013-7016; and Turnet et al. (1996) J. Biol. Chem. 271: 8966-8970). Below are examples of specific embodiments for carrying out the present invention. The examples are offered for illustrative purposes only, and are not intended to limit the scope of the present invention in any way.

Example 1 Identification of a Human cDNA Sequence Which Probably Encodes the Polypeptide P2Xg_. The predicted amino acid sequence of the rat P2X3 receptor (NCBI sequence l.D. number 1103623) was used to search the sequences Human DNA that would code for similar polypeptides. The database search tool TBLASTN (Altschul (1993J J.

Mol. Evol. 36: 290-300), which allows questioning the nucleotide database with a protein sequence by dynamically transferring the DNA sequences in all six possible reading frames. A fa database search of Genbank sequence tagged sites (STS) revealed a human genomic fragment, 229 base pairs in length, containing an open reading frame that would be predicted to encode a polypeptide that has a high degree of of homology for a region of the rat P2X3 receptor. The sequence deposited for this fragment (access number G03901 Genbank) was as follows: CCCGAATCGG TGGACTGC CTCCACTGTG GTC GGTCGC TGGGGTACAC TG GGTTGGTC AAAGCCGCGA TTTTCAGTGT AGTCTCATTC ACUTGNAGGC GAAAGAGCTG GTGTTGTCAA GT C GACTA TGGGCAATGT CC TT TTTTGT GACCCCA GACAGACTCA GCAGTGGGCG CCCATGACCT AGTCA GAGG GGAGCCAGGA CATC GTG G ATCCCAAGG (SBQ ID NO: l)? where "N" represents any of the bases A, T, G and C.

Example 2 Identification of the 5 'End of P2X * cDNA Based on the sequence of G03901, primers were designed for use in reverse transcription polymerase chain reaction (RT-PCR) methods in an effort to isolate the open reading frame intact for this receiver. The primaries used in the reactions described below were as follows: Primary 1s (SEQ ID NO: 2): d-TTTACCAACCCAGTGTACCC-S 'Primary 2s (SEQ ID NO: 3): 5'-ACCACAGTGGAGAAGCAGTC-3' Primary 3as (SEQ ID NO: 4): 5'-GAATCGGTGGACTGCTTCTC-3 'Primary 4as (SEQ ID NO: 5): 5'-CGATTTTCAGTGTAGTCTCATTC-3' Primary 5as (SEQ ID NO: 6): 5'-GGGGTACACTGGGTTGGTAA -3 '5' RACE Primary Anchor (SEQ ID NO.7): d-CUACUACUACUAGGCCACGCGTCGACTAGTACGGGHGGGI IGGGIIG-3 '(where "U" represents uracil e "I" represents inosine) Primary Universal Adapter (SEQ ID NO: 8) : d'-CUACUACUACUAGGCCACGCGTCGACTAGTAC * 3 'Primary Adapter (SEQ ID NO: 9): 5'-GGCCACGCGTCGACTAGTACTTTTTTTTTTTTTTTTT-3' Primary Universal Condensed Adapter (SEQ ID NO: 10): 5'-GGCCACGCGTCGACTAGTAC-3 'Primary 5'hP2X3 (SEQ ID NO: 1 1): d'-CACCATGAACTGCATATCCGACTTC-3 'Primary 3'hP2X3 (SEQ ID NO: 12): d'-CTAGTGGCCTATGGAGAAGGC-3' To identify the 5 'end of the cDNA that is derived from the genomic region of which the sequence G03901 is a part, the RACE technique (Rapid Amplification of cDNA Ends) (Frohman et al. (1988), Proc. Nati. Acad. Sci. USA 85: 8998-9002). The extension of the cDNA identified through the RT-PCR step was performed using the 5 'RACE ™ reagent system (Life Technologies, Gaithersburg, MD). A microgram of poly A + RNA derived from human pituitary gland tissue (Cat. # 65894-1, Lot # 6080167; Clontech Laboratories, Palo Alto, CA) in a reaction using reagents provided in the package as described; 1 μl (1 μg) of RNA was combined with 3 μl (3 pmol) of 3a primary and 11 μl of RNase-free water (water treated with diethylpyrocarbonate, or DEPC) and heated at 70 ° C for 10 minutes followed by 1 minute on ice. A mixture of 2.5 μl of 10x reaction buffer (200 mM tris-HCl pH 8.4, 500 mM KCl) was added. 3 μl 25 mM MgCl2, 1 μl 10 mM dNTP, and 2.5 μt 0.1 M DTT. The mixture was incubated at 42 ° C for 2 minutes after which 1 μl of Superscript ll ™ reverse transcriptase (Life Technologies) was added. The reaction was incubated for an additional 30 minutes at 42 ° C, 15 minutes at 70 ° C, and on ice for 1 minute. One microliter of RNase H (2 units) was added and incubated at 55 ° C for 20 minutes. The cDNA was purified using the GlassMax ™ columns included in the package. The cDNA was eluted from the column in 50 μl of distilled water (dH2O), lyophilized, and resuspended in 21 μl of dH = O. The cDNA was monitored in the following reaction: 7.5 μl of dH2O, 2.5 μl of reaction buffer (200 mM tris-HCl pH 8.4, 500 mM KCi), 1.5 μl 25 M MgCl2, 2.5 μf 2 mM dCTP, and 10 μl of the cDNA were incubated at 94 ° C for 3 minutes, then 1 minute on ice, followed by 10 minutes at 37 ° C. Finally, the mixture was incubated at 70 ° C for 10 minutes and then placed on ice. The PCR amplification of the cDNA was performed in the following steps: 5 μl of the cDNA were included in a reaction that also contained a mixture of 5 μl of 10x GeneAmp ™ PCR buffer (Perkin Elmer, Foster City, CA) (500 M KCl , 100 mM tris-HCl pH 8.3, 15 mM MgCl2, and 0.01% (w / v) gelatin), 1 μl 10 mM dNTP, 1 μl (10 pmol) of primary Anchor, 1 μl (10 pmol) of primary das, and 3d μl of dH2O. The reaction was heated to 9d ° C for 1 minute, then maintained at 80 ° C for 2 minutes, during which time O.d μl (2.d units) of Amplitaq ™ d polymerase (Perkin-Elmer) was added. The reaction was recirculated 3d times under these conditions: 94 ° C for 1d seconds, 62 ° C for 20 seconds, and 72 ° C for 1 minute. After amplification, the reaction products were purified using the QiaQuick ™ PCR product purification system (Qiagen, Inc., Chtsworth CA) according to the manufacturer's instructions. The products were eluted from the columns with dO μl TE buffer (10 mM tris, 1 mM EDTA pH 8.0), and one microliter of the eluent was used as a hardened DNA in a PCR reaction to increase specific product levels for isolation. subsequent Re-amplification also included: dll of 10x GeneAmp ™ PCR buffer, 1 μl 10 mM dNTP mixture, 1 μl (10 pmol) of Universal Amplification primary, 1 μl (10 pmol) of primary 4a, and 40.6 μl of dH2O. The reaction was heated to 9d ° C for 1 minute, then maintained at 80 ° C during which 0.5 μl (2.5 0 units) of Amplitaq ™ polymerase was added. The reaction was recycled 35 times under these conditions: 94 ° C for 1 d seconds, 60 ° C for 20 seconds, and 72 ° C for 1 minute. The amplification products were analyzed via 0.8% agarose gel electrophoresis and a predominant product of approximately 1.3 kilobase pairs of d length was detected. This product was removed from the gel and purified via the QiaQuick ™ purification system. The product was eluted from the column with dO μl of dH = O and lyophilized at 10 μl volume. 3 microliters of the resulting DNA was used in a lCR reaction with pCR 2.1 vector (Invitrogen, Carlsbad, CA) incubated at 14 ° C d overnight. The ligation products were used to transform E. coli from the cloning package using standard manufacturer protocols. The insert sizes of the resulting clones were determined using EcoRI digestions of the plasmids and clones containing inserts of the approximate size of the PCR product were 0 sequenced using fluorescent dye terminator reagents (Prism ™, Perkin Elmer Applied Biosystems Division, Foster City , CA) and an Applied Biosystems DNA sequencer model 373. The sequence of the d'RACE product including the EcoRI sites of the pCR 2.1 vector is shown in FIG. 1 (SEQ ID NO: 13). The sequences of the 5 amplifiers (Universal Amplification Primer and the complement to Primer 4as) are underlined.

EXAMPLE 3 Identification of the 3 'End det P2X3 cDNA 0 To identify the sequence surrounding the termination codon of the open reading frame encoding the human P2X3 receptor, the Life Technologies 3'RACE ™ System was used with primers designed for STS G03901. Poly A + RNA (dOO nanograms) derived from pituitary gland tissue (see Example 2, above) was used in the reaction as follows: RNA and 10 picomoles of Primary Adapter were combined in a final volume of 12 μl of dH2O. This mixture was heated at 70 ° C for 10 minutes and cooled on ice for 1 minute. The following components were added: 2 μl of 10x PCR buffer (200 mM of tris-HCl pH 8.4, dOO mM of KCl), 2 μl of 26 mM MgCl 2, 1 μl of 10 mM of dNTP mixture, and 2 μL of 0.1 M dithiothreitol. The reaction was equilibrated at 42 ° C for 2 minutes after which 1 μl (200 units) of Superscript II ™ reverse transcriptase was added and incubation was continued at 42 ° C for 60 minutes. The reaction was terminated by incubation at 70 ° C for 15 minutes and cooled on ice. 0 RNase H (1 μl, 2 units) was added and the mixture was incubated for 20 minutes at 37 ° C, and then stored on ice. Amplification of the 3 'end of the cDNA P2X3 was carried out in the following reactions: 2 μl of the first cDNA filament synthesized before in a PCR mixture was used, also including 5 μl of 10 × GeneAmp ™ PCR buffer, 1 μl dNTPs 10 mM 1 μl (10 picomoles) of Primary 1 s, 1 μl (10 picomoles of Universal Condensed Amplification Primary (AUAP) and 39. d μl of dHjO.) The reaction was heated at 9d ° C for 1 minute, then maintained at 80 ° C for 2 minutes, during which Od μl (2.6 units) of 0 Amplitaq ™ polymerase was added.The reaction was recycled 35 times under these conditions: 94 ° C for 16 seconds, 64 ° C for 20 seconds, and 72 ° C for 2 minutes After recycling, the reaction was incubated for 10 minutes at 70 ° C and stored at 4 ° C. After the amplification, the reaction products were purified using the PCR product purification system. QiaQuick ™ according to the instructions of the f The products were eluted from the columns with 60 μl of TE buffer (10 mM tris, 0.1 mM EDTA pH 8.0), and one microliter of the eluent was used as a hardened DNA in a PCR reaction to increase specific product levels for isolation subsequent Re-amplification also included: 5 μl of 10x GeneAmp ™ PCR buffer, 1 μl of 10 mM dNTP mixture, 1 μl (10 pmol) of AUAP, 1 μl (10 pmol) of primary 2s, and 40.5 μf of dH2O. The reaction was heated at 95 ° C for 1 minute, then maintained at 80 ° C during which 0.5 μt (2.5 units) of Amptitaq ™ potimerase was added. The reaction was recycled 36 times under these conditions: 94 ° C for 16 seconds, 54 ° C for 20 seconds, and 72 ° C for 2 minutes. The amplification products were analyzed via 0.8% agarose gel electrophoresis and a predominant product of approximately 700 base pairs in length was detected. This product was removed from the gel and purified via the QiaQuick ™ purification system. The product was eluted from the column with 50 μl of dH2O and l? Ofilized to 10 μl of volume. 3 microliters of the above DNA was used in a ligation reaction with pCR 2.1 vector (Invitrogen) incubated at 15 ° C for 3.5 hours. The ligation products were used to transform E. coli from the cloning package. The insert sizes of the resulting clones were determined using EcoRI digestions of the plasmids and clones containing inserts of the approximate size of the PCR product were sequenced using fluorescent dye terminator reagents (Prism ™, Applied Biosystems) and a DNA sequencer from Applied Biosystems 373. The sequence of the 3'RACE product including the EcoRI sites of the pCR 2.1 vector is shown in Figure 2 (SEQ ID NO: 14), in which the sequences of the amplifiers (AUAP and the complement to Primary 2s) they are underlined Example 4 Isolation of cDNA containing the Intact Open Reading Frame of P2X? Human Using information from the sequence surrounding the initiation and termination codons of the human P2X3 message, primers of oligonucleotide were designed and synthesized to enable the RT-PCR for intact open reading frame of the mRNA. The sequences of these primaries, designated d'hP2X3 and 3'hP2X3, are shown above. The PCR amplification was performed in a portion (2 ul) of the pituitary gland cDNA described in Example 3. A thermostable polymerase was used for reading (Cloned Pfu DNA Potymerase, Strategene, La Jolfa, CA) in amplification to ensure high fidelity amplification. The reaction mixture consisted of 2 μl of cDNA, dμl of 10x cloned Pfu polymerase reaction buffer (200 mM tris-HCl (pH 8.8), 100 M KCi, 100 mM (NH 4) 2 SO 4, 20 mM MgSO 4, Triton X- 100 to 1%, 1 mg / ml of nuclease-free bovine serum albumin), 1 μl of dNTP mixture, 1 μl (10 picomoles) of Primary 5'hP2X3, 1 μl (10 picomoles) of Primary 3'hP2X3, and 39.5 μl of dH2O. The reaction was heated at 95 ° C for 1 minute, then kept at 80 ° C for 2 minutes, during which time O.d μl (1.26 units) of cloned Pfu polymerase was added. The reaction was recycled 36 times under the following conditions: 94 ° C for 20 seconds, 62 ° C for 20 d seconds, and 72 ° C for 3.6 minutes. After recycling, the reaction was incubated for 10 minutes at 70 ° C. The reaction products were separated on an 0.8% agarose gel and an approximately 1.2 kilobase product was removed and purified via the QiaQuick ™ gel purification system. . The DNA was either eluted with 50 μl of dH2O, lyophilized and resuspended in 10 μl of dH2O. A microliter of this DNA was used in a re-amplification reaction which also included 5 μl of 10x Pfu reaction buffer, 1 μl of dNTP mixture, 1 μl (10 pmol) of Primary of hP2X3, 1 μl (10 pmol) of primary 3'hP2X3, and 40.5 μl of dH2O. The reaction was heated at 96 ° C for 1 minute, then maintained at 80 ° C for 2 minutes, during which O.d μl (1.25 units) of cloned Pfu polymerase was added. The reaction was recycled 15 times under the following conditions: 94 ° C for 20 seconds, 52 ° C for 20 seconds, and 72 ° C for 3.5 hours. After recycling, the reaction was incubated 0 for 10 minutes at 70 ° C. The reaction products were separated on a 0.8% agarose gel and the 1.2 kilobase product was removed and purified via the QiaQuick ™ gel purification system. . The DNA was eluted with 50 μl of dH2O, lyophilized and resuspended in 5 μl of dHaO. 3 microliters of the purified PCR product was used in a ligation reaction using the pCRScript ™ cloning system (Stratagene) which also included 5 μl (5 ng) of the pCRScript ™ Amp SK (+) vector, 1 μl of buffer pCRScript ™ 10x reaction, 0.5 μm 10 mM ATP, 1 μl (5 units) Srf i restriction enzyme, 1 μl (4 units of T4 DNA ligase, and 3 μl dH2O) The reaction mixture was incubated at room temperature for 1 hour, then at 65 ° C for 10 minutes One microliter of this reaction product was used to transform blue XL-2 ultracompetent cells (Stratagene) according to the manufacturer's standard protocols. restriction analysis and sequenced using fluorescent dye terminator reagents (Prism, Applied Biosystems) and a DNA sequencer from Applied Biosystems model 310. The sequence of intact open reading frame is shown in Figure 3 (SEQ ID NO: 1 d) Figure 4 shows a comparison of the predicted sequence of the human P2X3 proteins of the present invention (SEQ ID NO: 16) with that of the corresponding rat polypeptide (SEQ ID NO: 17) .

Example 5 Expression and Electrophysiological Analysis of Recombinant P2X3 Receptors in Xenopus Oocytes Prepared and injected with recipient DNA of the present invention, Xenopus laevis oocytes, and receptor responses were measured using 2-electrode voltage clamps, according to previously described procedures (Briggs et al. (1995), 5 supra). The oocytes were maintained at 17-18 ° C in normal Barth's solution (NaCl, 90 M, 1 mM KCl, 0.66 mM NaNO3, 0.74 mM CaCl2, 0.82 mM MgCl2, 2.4 mM NaHCO3, 2.6 mM sodium pyruvate, and Na N- (2-Hydroxy-ethyl) -piperazine-N '- (2-etansutfonic acid) ("HEPES"), final pH 7.66) containing 100 μg / ml of gentamicin. The responses were measured at a clamping potential of -60 mV in modified Barth's solution containing 10 mM BaCI2 and lacking CaCl2 and MgCl2 (final pH 7.4). However, in some experiments, the cell potential was intentionally varied in order to determine the current-voltage response relationship. The agonist was briefly applied using a computer controlled solenoid valve and a push / pull applicator placed within 200-400 μm det oocyte. Responses were recorded by computer in synchrony with agonist application. Antagonists with agonist were included in the push / pull applicator and were applied to the bath by superfusion for at least 3 minutes before the application of agonist. The responses were quantified by measuring the amplitude of the peak. The DNA for injection into oocytes was the P2X3 insert of pCDNA3.1 prepared as described in Example 2. The clone was grown and prepared on a large scale using QlAgen maxiprep DNA preparation system according to the instructions of maker. The DNA was precipitated with ethanol and resuspended in TE buffer. For the production of RNA the construction of P2X3-pCDNA3.1 was linearized by digestion with the restriction enzyme NotI and P2X3 messenger RNA was produced using the T7 promoter in this vector and the in vitro transcription package Ambíon mMessage mMachine ™ according to with the manufacturer's instructions. For functional analysis of human P2X3 receptors, they were injected ng of P2X3 human DNA prepared as described above to the Xenopus oocyte nucleus. The oocytes were incubated in normal Barth's solution containing 100 μg / ml of gentamicin for 2-7 days after injection. The response to 10 μM of ATP was then recorded. The results of the above expression and analysis show that the receptors of the present invention are functional. The oocytes injected with P2X3 of human DNA responded to the extracellular application of ATP exhibiting a mixed conductance cation current (100-6000 nA). Oocytes injected with an appropriate amount of water did not respond to ATP. An approximate ATP ECD of 0.7 μM was obtained from concentration-response relationships (0.01-1000 μM) of these oocytes. The current-voltage relationships induced by ATP were also recorded from these oocytes. This revealed an inversion potential of approximately zero mV, with pronounced inward rectification recorded in negative membrane potentials. Another P2X receptor agonist, a, β-methylene-ATP, eliminated peak currents similar to those evoked by ATP, although it was slightly less potent (EC50 = 2.1 μM). The application of a third P2X receptor agonist, 2-methyptide-ATP, was slightly more potent (EC50 = 0.4 μM) than either ATP or a, β-methylene-ATP. Functional antagonism of responses was determined by application of P2X receptor antagonists not specific for suramin or pyridoxat-phosphate-6-azophenyl-2, 4'-disulfonic acid (PPADS). Both antagonists produced a complete block of induced currents (0.3 μM) of ATP, with suramine exhibiting increased potency (ICso = 0.3 μM) in relation to PPADS (ICSQ = 1 μM). In summary, the injection of P2X3 human DNA receptor into Xenopus oocytes resulted in expression of functional P2X3 receptors on the cell surface, and these receptors function as non-specific cation channels with ligand gate. These receptors respond to extracellular P2 receptor agonists with a power range of the order of 2-methylthio-ATP > ATP > a, ß-metiIen-ATP. They also exhibit inward rectification and are blocked by both P2 PPADS receptor antagonists and suramin.

LIST OF SEQUENCES < 110 > Abbott Laboratories Lynch, Kevin J. Burgard, Edward C. Van Biesen, T. < 120 > Nucleic Acids that Encode a P2X3 Functional Purinoreceptor Human and Methods of Production and Use of the same. < 130 > 6293. PC.01 < 1d0 > US 09 / 008,526 < 151 > 1998-01 -16 < 160 > 17 < 170 > FastSEQ for Windows Version 3.0 < 210 > 1 < 211 > 229 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Oligonucleotide for PCR < 220 > < 221 > modified base < 222 > (93) ... (93) < 223 > n = a og or co t / u, unknown, or other < 220 > < 221 > modified base < 222 > (96) ... (96) < 223 > n = a or g or co t / u, unknown, or other < 400 > 1 cccgaatcgg tggactgctt ctccactgtg gtctggtcgc tggggtacac tgggttggtc 60 aaagccgcga ttttcagtgt agtcteattc acntgnaggc gaaagagctg gtgttgtcaa 120 gttctgacta tgggcaatgt cctcttttgt gaccecattt gacagactca gcagtgggcg 180 cccatgacct agtcatgagg ggagccagga catctgtgtg atcccaagg 229 < 210 > 2 < 211 > 20 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Sequenced Primary < 400 > 2 tttaccaacc cagtgtaccc < 210 > 3 < 21 1 > 20 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Sequenced Primary < 400 > 3 accacagtgg agaagcagtc 20 < 210 > 4 < 21 1 > 20 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Sequenced Primary < 400 > 4 gaatcggtgg actgcttctc 20 < 210 > 5 < 211 > 23 < 212 > DNA < 213 > Sequence, Artificial < 220 > < 223 > Sequenced Primary < 400 > 5 cgattttcag tgtagtctca ttc 23 < 210 > 6 < 211 > 20 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Sequenced Primary < 400 > 6 ggggtacact gggttggtaa 20 < 210 > 7 < 211 > 48 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > d'Anchor Primary RACE < 220 > < 221 > modified base < 222 > (36) ... (37) < 223 > n = a og or co t / u, unknown, or other < 220 > < 221 > modified base < 222 > (41) ... (42) < 223 > n = aogoco t / u, unknown, or other < 220 > < 221 > modified base < 222 > (46) ... (47) < 223 > n = a or g or c or t / u, unknown, or other < 400 > 7 cuacuacuac uaggccacgc gtcgactagt acgggnnggg nngggnng 48 < 210 > 8 < 21 1 > 32 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Primary Universal Adapter < 400 > 8 cuacuacuac uaggccacgc gtcgactagt ac 32 < 210 > 9 < 21 1 > 37 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Primary Sequencing Adapter < 400 > 9 ggccacgcgt cgactagtac tttttttttt ttttttt 37 < 210 > 10 < 21 1 > 20 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Primary Universal Sequencing Adapter Condensed < 400 > 10 ggccacgcgt cgactagtac 20 < 210 > 11 < 211 > 2d < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Sequenced Primary < 400 > 11 caccatgaac tgcatatccg acttc 26 < 210 > 12 < 211 > 21 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Sequenced Primary < 400 > 12 ctagtggcct atggagaagg c 21 < 210 > 13 < 211 > 1272 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Sequenced Primary < 400 > 13 ctactactac taggccacgc gtcgactagt acgggggggg S339333"cc ggggacgacc 60 accacctacc tcctcaggct gcggcctcgc gagggccccg gcgegagagg acccccctct 120 sctgaggcca ccactgggec cscttctgag tgtcccctga gsastctctc agcatgaact 180 gcatatccga cttcttcacc tatgagacca ccaagtcggt agctggacca ggttgtgaaa 240 tcgggatcat caaccgagta gttcagettc tgatcatctc ctactttgta gggtgggttt 300 tcttgcacga gaaggcttac caggtacggg acacagccat taagtcctcg gtggtaacca 360 aggtgaaggg etccggactc tacaccaaca gagtcatgga tgtgtctgat tacgtgacgc 420 cacetcaggg cacctcggtc tttgtcatca tcaccaagat gattgttact gaaaatcaga 480 tgcaaggatt ctgcccagag agtgaggaga aafcaccgctg tgtafccagac agccagtgcg 540 ggcegagcg cttgccaggg atcctcactg gccgctgcgt gaactacagc tctgcgctcc 600 ggacct ^ tga tggtgccsca gatccagggc cggaggtgga cacagtggaa acgcccatca 660 tgatggaage tgagaacttc actattttca tcaagaacag catccgtttc cccctcttca 720 actttgagaa gggaaacctc cttcccaacc tgacagccag ggacatgaag acctgcegct 780 tccacccgga caaggaccct ttctacccca tcttgcgggt aggggacgtg gtcaagtttg 840 cggggcagga tttt gccaaa ctggcgcgca cggggggagt tctgggcatt aagatcggct 900 gg fcgtgcga cttggacaag gcctgggacc agtgcatccc caaatactcc ttcacccggc 960 tcgacagcgt ttctgagaaa agcagcgtgt ccccaggcta caacttcagg tttgccaagt 1020 actacaaaat ggaaaatggc agtgagtacc gcaccctcct gaaggctttt ggcatccgct 1080 tcgacgtget ggtatacggg aatgctggca agttcaacat catccccacc atcatcagct 1140 ctgtggoggc cfcttacttct gtgggagtgg gaactgttct ctgtgacatc atcctgctca 1200 acttcctcag gggggccgac cagtacaaag ccaagaagtt tgaggaggtg aatgagacta 1260 cactgaaaat eg 1272 < 210 > 14 < 211 > 706 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Sequenced Primary < 40Q > 14 accacagtgg agaagcagtc caccgattcg ggggccttct ccataggsca ctagggcctc 60 tttceagggc cccacactca caaaggctcc aggcctcscc acagaggacc ctgcctgagc 120 * »gggggcat gggagggaag aggggctetc atttctgctg ctcattccat gagcatagct 180 gggaeccaag tgtctgggcc tccgactgct ccagcagaca ggcagtgctc cctgctgaga 240 ccccagtctc accttcactc cttgcctggc cccatctgct tcctaggacc cctggggcag 300 gagcacctga gccatcccct tcccaaagag tagagattat aatgtaggac agatggccac 360 aagggcctac caagtgccag gcactttcac acacgttatc tcatttaatc cttagaafcaa 420 tcctatgagg tagatattag tttcccttgt tttgaagata aaccaaggct cagagagact 480 gagtcatttg ccccaggcca gatagccagg atgtgagaga gctgggattt gaacgtccgt 540 ccatcgccca ctgactaact caccccatga gagaagatga actcccaggg tccatcagcc 600 ctgctgcttc agccgcctcc accctgacgg tgattcggtt aaaaaaaaaa aaaaaaaaaa 660 aataaagagt aagccccaaa aaaaaagtac tagtcgacgc gtggcc 706 < 210 > 15 < 21 1 > 1243 < 212 > DNA < 213 > Homo Sapiens < 400 > 15 gaattcctgc agcccggggg gatccgcccc accatgaact gcatatccga cttcttcacc 60 tatgagacca ccaagtcggt ggttgtgaag agctggacca tcgggatcat caaccgagta 120 gttcagcttc tgatcatctc ctactttgta gggtgggttt tcttgcacga gaaggcttac 180 caggtacggg acacagccat tgagtcctcg gtggtaacca aggtgaaggg ctccggactc 240 gagtcatgga tacgccaaca tgtgtctgat tacgtgacgc cacctcaggg cacctcggte 300 tttgtcatca tcaccaagat gattgttact gaaaatcaga tgcaaggatt ctgcccagag 360 agtgaggaga aataccgctg tgtatcagac agccagtgcg ggcctgagcg cttgccaggt 420 ggggggatcc tcactggccg ctgcgtgaac tacagctctg tgctccggac ctgtgagatc 480 cagggctggt gccccacgga ggtggacaca gtggaaacgc ccatcatgat ggaagctgag 540 ttttcatcaa aacttcacta gaacagcatc cgtttccccc tcttcaactt tgagaaggga 600 aacctccttc ccaacctgac agccagggac atgaagacct gccgcttcca cccggacaag 660 gaccctttct gccccatctt gcgggtaggg gacgtggtca agtttgcggg acaggatttt 720 gccaaactgg cgcgcasggg gggagttctg ggcattaaga tcggctgggt gtgcgacttg 780 gacaaggcct gggaccagtg catccccaaa tactccttca cagcgtttct cccggctcga 840 gagaaaagca gcgtgtc ccc aggctacaac ttcaggtttg ccaagtacta caaaatggaa 900 aatggcagtg agtaccgcac cctcctgaag tccgcttcga gcttttggca cgtgctggta 960 tacgggaatg ctggcaagtt caacatcatc cccaccatca tcagctctgt ggcggccttt 1020 acttctgtgg gagtgggaac tgttctctgt gacatcatcc tgctcaactt cctcaagggg 1080 gccgaccagt acaaagccaa gaagtttgag gaggtgaatg agastacgct gaaaatcgcg 1140 acccagtgta gctttgacca ccccagcgac cagaccacag cggagaagca gtccaccgat 1200 tcgggggcct tctccatagg ccactagggg ctagagcggc cgc 1243 < 210 > 16 < 211 > 397 < 212 > PRT < 213 > Homo Sapiens < 400 > 16 Met Asn Cis He Ser Asp Fe Faith Tr Tir GIu Tr Tr Lis Ser Val 5 10 15 Val Val Lis Ser Trp Tr lie Gli lie lie Asn Arg Val Val Gln Leu 20 25 30 Leu He He Ser Tir Fe Val Gli Trp Val Fe Leu His Glu Us Wing 35 40 45 Tir Gln Val Arg Asp Tr Wing He Glu Ser Val Val Tr Lis Val 60 dd 60 Lis Gli Ser Gli Leu Tir Wing Asn Arg Val Met Asp Val Ser Asp Tir 66 70 76 80 Val Tr Pro Pro Gln Gli Tr Ser Vat Fe Val He He Tr Tr Met Met 85 90 95 He Vat Tr Gfu Asn Gln Met Gln Gli Faith Cis Pro Glu Ser Glu Glu 100 105 110 Lis Tir Arg Cis Val Ser Asp Ser Gln Cis Gli Pro Gtu Arg Leu Pro 115 120 125 Gli Gli Gli He Leu Tr Gli Arg Cis Val Asn Tir Being Ser Vat Leu 130 135 140 Arg Tr Cis Gtu He Gln Gli Trp Cis Pro Tr Glu Val Asp Tr Val 145 150 155 160 Glu Tr Pro He Met Met Glu Ala Glu Asn Faith Tr He Faith He Lis 165 170 175 Asn Ser He Arg Faith Pro Leu Faith Asn Faith Glu Lis Gti Asn Leu Leu 180 185 190 Pro Asn Leu Tr Ala Arg Asp Met Lis Tr Cis Arg Fe His Pro Asp 195 200 205 Lis Asp Pro Fe Cis Pro He Leu Arg Val Gli Asp Val Val Lis Fe 210 215 220 Wing Gli Gln Asp Faith Ala Lis Leu Wing Arg Tr Gli Gli Vaf Leu Gli 225 230 235 240 He Lis He GH Trp Val Cis Asp Leu Asp Lis Ala Trp Asp Glen Cis 245 250 256 He Pro Lis Tir Ser Fe Tr Arg Leu Asp Ser Val Ser Glu Lis Ser 260 265 270 Ser Val Ser Pro Gli Tir Asn Faith Arg Faith Ala Lis Tir Tir Met Met 275 280 285 Glu Asn Gli Ser Glu Tir Arg Tr Leu Leu Lis Wing Faith Oli He Arg 290 295 300 Faith Asp Val Leu Val Tir Gli Asn Ala Gli Lis Fe Asn He He Pro 30d 310 316 320 Tr He He Ser Ser Val Ala Ala Fe Tr Tr Ser Val Gli Val Gli Tr 326 330 336 Val Leu Cis Asp He He Leu Leu Asn Faith Leu Lis Gli Wing Asp Gln 340 34d 360 Tir Lis Ala Lis Lis Fe Glu Glu Val Asn Glu Tr Tr Leu Lis He 365 360 365 Wing Ala Leu Tr Asn Pro Val Tir Pro As Asp Gl Tr Tr Ala Glu 370 375 380 Lis Gln Ser Tr Asp Ser Gli Ala Fe Ser He Gli His 385 390 395 < 210 > 17 < 21 1 > 397 < 212 > PRT < 213 > Rattus rattus < 400 > 17 Met Asn Cis lie Ser Asp Faith Fe Tr Tir Glu Tr Tr Lis Ser Val 1 5 10 15 Val Val Lis Ser Trp Tr He Gli lie lie Asn Arg Val Val Gln Leu 20 25 30 Leu He He Ser Tir Fe Val Gli Trp Val Fe Leu Hís Glu Lis Ala 35 40 45 Tir Gln Val Arg Asp Tr Ala lie Glu Ser Val Val Tr Lis Val 50 56 60 Lis Gli Ser Gli Leu Tir Ala Asn Arg Val Met Asp Val Ser Asp Tir 65 70 75 80 Val Tr Pro Pro Gln Gli Tr Val Val Ser He He Tr Tr Lis lie 85 90 95 He Val Tr Glu Asn Gln Met Gln Gli Faith Cis Pro Glu Asn Glu Glu 100 105 1 10 Lis Tir Arg Cis Val Ser Asp Ser GIn Cis Gli Pro Glu Arg Fe Pro 115 120 125 Gl? Gii Gli He Leu Tr Gli Arg Cis Val Asn Tir Ser Ser Val Leu 130 13d 140 Arg Tr Cis Glu He Gln Gli Trp Cis Pro Tr Glu Val Asp Tr Val 146 15 0 165 160 Glu Met Pro Met Met Met Glu Ala Glu Asn Fe Tr Met Fe lie Lis 165 170 175 Asn Be He Arg Faith Pro Leu Faith Asn Faith Glu Lis Gli Asn Leu Leu 180 185 190 Pro Asn Leu Tr Asp Lis Asp He Lis Arg Cis Arg Fe His Pro Glu 195 200 205 Lis Wing Pro Fe Cis Pro He Leu Arg Val Gli Asp Val Val Lis Fe 210 215 220 Wing Gli Gln Asp Faith Wing Lis Leu Wing Arg Tr Gli Gli Val Leu Gli 225 230 235 240 He Lis He Gli Trp Val Cis Asp Leu Asp Lis Wing Trp Asp Gln Cis 245 250 25d He Pro Lis Tir Ser Fe Tr Arg Leu Asp Gli Val Ser Glu Lis Ser 260 265 270 Ser Val Ser Pro Gli Tir Asn Faith Arg Fe Ala Lis Tir Tir Lis Met 275 280 285 Glu Asn Gli Ser Glu Tir Arg Tr Leu Leu Lis Wing Faith Gli He Arg 290 295 300 Faith Asp Val Leu Val Tir Gii Asn Wing Gli Lis Fe Asn He He Pro 305 310 315 320 Tr He He Ser Ser Val Ala Ala Fe Tr Ser Val Gli Val Gii Tr 325 330 335 Val Leu Cis Asp He He Leu Leu Asn Faith Leu Lis Gli Wing Asp His 340 345 350 Tir Lis Ala Ar Lis Fe Glu Glu Ala Tr Val Glu 370 375 380 Lis Gln Ser Tr Asp Ser Gli Ala Tir Ser He Gli His 385 390 395

Claims (39)

1. CLAIMS 1. An isolated polynucleotide that encodes a human P2X3 receptor polypeptide or a degenerate variant thereof.
2. 2. A polynucleotide according to claim 1, wherein the polynucleotide is a polideoxyribonucleotide (DNA).
3. 3. A polynucleotide according to claim 1, wherein the polynucleotide is a polyribonucleotide (RNA).
4. 4. A polynucleotide according to claim 2, wherein the DNA comprises the sequence SEQ ID NO: 15. d.
5. A host cell comprising a polynucleotide according to claim 1 or claim 4.
6. 6. A host cell according to claim 5, wherein said cell is selected from the group consisting of a bacterial cell, a mammalian cell, a yeast cell and an amphibian cell.
7. 7. A host cell according to claim 6, wherein the cell is an amphibian cell.
8. 8. A host cell according to claim 6, wherein the cell is a mammalian cell.
9. 9. An expression vector comprising a polynucleotide according to claim 1 operably linked to control sequences that direct the transcription of the poolinucleotide, whereby the polynucleotide is expressed in a host cell.
10. 10. An expression vector according to claim 9, wherein the human P2X3 receptor polypeptide comprises the sequence d 63 amino acids of SEQ ID NO: 16.
11. 11. A host cell comprising an expression vector according to claim 9.
12. 12. A host cell according to claim 1, wherein the cell is selected from the group consisting of a bacterial cell, a mammalian cell, a yeast cell and an amphibian cell.
13. 13. A host cell according to claim 12, wherein the cell is an amphibian cell.
14. 14. A host cell according to claim 12, wherein the cell is a mammalian cell. 16.
15. A host cell comprising the expression vector of claim 10.
16. 16. A host cell according to claim 1d, wherein the cell is selected from the group consisting of a bacterial cell, a mammalian cell, a cell of yeast and an amphibian cell.
17. 17. A host cell according to claim 16, wherein the cell is an amphibian cell.
18. 18. A host cell according to claim 16, wherein the cell is a mammalian cell.
19. 19. A method for producing a human P2X3 receptor polypeptide, the method comprising the steps of: (a) culturing a host cell according to claim 1 under conditions that allow production of the polypeptide; and (b) recovering the polypeptide.
20. 20. A method for producing a human P2X3 receptor polypeptide, the method comprising the steps of: (a) culturing a host cell according to claim 15 under conditions that allow production of the polypeptide; and (b) recovering the polypeptide.
21. 21. An isolated and purified human P2X3 receptor polypeptide, wherein the human P2X3 receptor comprises the amino acid sequence of SEQ ID NO: 16.
22. 22. A method for identifying compounds that modulate P2X receptor activity, the method comprising the steps of: (a) providing a cell that expresses a P2X receptor comprising a human P2X3 polypeptide; (b) mixing a test compound with the P2X receptor; and (c) measuring either: (i) the effect of the test compound on the activation of the P2X receptor or the expression of the P2X receptor cell, or (ii) the binding of the test compound to the cell or receptor of P2X.
23. 23. A method according to claim 22 wherein the host cell is selected from the group consisting of a bacterial cell, a mammalian cell, a yeast cell and an amphibian cell.
24. 24. A method according to claim 22 wherein said measuring step (c) (ii) is performed by measuring a signal generated by a detectable portion.
25. 25, A method according to claim 24 wherein said detectable portion is selected from the group consisting of a fluorescent label, a radio label, a chemiluminescent label and an enzyme.
26. 26. A method according to claim 22 wherein said measurement step (c) (i) is performed by measuring a signal generated by a radiolabelled ion, a chromogenic reagent, a fluorescent probe or an electric current.
27. 27. A method according to claim 23, wherein the host cell is a mammalian cell.
28. 28. A method according to claim 23, wherein the host cell is an amphibian cell.
29. 29. A method according to claim 22, wherein the human P2X3 receptor polypeptide comprises the amino acid sequence of SEQ ID NO: 16.
30. 30. A method for detecting an obsective polynucleotide of a P2X3 receptor in a test sample, the method comprising the steps of: (a) contacting the target polynucleotide with at least one specific P2X3 receptor polynucleotide probe or a complement thereof to form a target probe complex; and (b) detecting the presence of the target probe complex in the test sample.
31. 31. A method for detecting human P2X3 receptor mRNA cDNA in a test sample, the method comprising the steps of: (a) performing reverse transcription in order to produce cDNA; (b) amplifying the cDNA obtained from step (a); and (c) detecting the presence of the human P2X3 receptor in the test sample.
32. 32. A method according to claim 31, wherein said detection step (c) comprises using a detectable portion capable of generating a measurable signal.
33. 33. A purified polynucleotide or a fragment thereof derived from human P2X3 receptor and capable of selectively hybridizing to a nucleic acid encoding a human P2X3 receptor polypeptide, wherein said polynucleotide comprises the sequence of SEQ ID NO: 15 or a portion of ia
34. 34. A purified polynucleotide according to the claim 33, wherein the polynucleotide is produced by recombinant techniques.
35. 35. A polypeptide encoded by a human P2X3 receptor polynucleotide wherein said polynucleotide comprises the amino acid sequence of SEQ ID NO: 16 or a portion thereof.
36. 36. A polypeptide according to claim 35 produced by recombinant techniques.
37. 37. A polypeptide according to claim 35 produced by synthetic techniques.
38. 38. A monoclonal antibody that specifically binds to the human P2X3 receptor comprising the amino acid sequence of SEQ ID NO: 16 or an immunoreactive fragment thereof.
39. 39. A method for detecting human P2X3 receptor in a test sample, the method comprising the steps of: (a) contacting the test sample with an antibody or a fragment thereof that specifically binds to the human P2X3 receptor, for a time and under conditions sufficient for the formation of a resulting complex; and (b) detecting the resulting complex containing the antibody, wherein said antibody specifically binds to the human P2X3 receptor amino acid comprising the amino acid sequence of SEQ ID NO: 16 or a fragment thereof.