WO1999036539A1

WO1999036539A1 - Nucleic acids encoding a functional human purinoreceptor p2x3 and methods of production and use thereof

Info

Publication number: WO1999036539A1
Application number: PCT/US1999/000963
Authority: WO
Inventors: Kevin J. Lynch; Tim Van Biesen; Edward C. Burgard
Original assignee: Abbott Laboratories
Priority date: 1998-01-16
Filing date: 1999-01-15
Publication date: 1999-07-22
Also published as: CA2317449A1; EP1045903A1

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 ENCODING A FUNCTIONAL HUMAN PURINORECEPTOR P2X,_^ AND METHODS OF PRODUCTION AND USE THEREOF

Technical Field

The invention relates generally to receptor proteins and to DNA and RNA molecules encoding therefor. In particular, the invention relates to a nucleic acid that encodes a human receptor P2X₃. The invention also relates to methods of using the P2X encoded thereby to identify compounds that interact with it.

Background of the Invention

P2 receptors have been generally categorized as either metabotropic nucleotide receptors or ionotropic receptors for extracellular nucleotides. Metabotropic nucleotide receptors (usually designated P2Y or P2Y_n, where "n" is a subscript integer indicating subtype) are believed to differ from ionotropic receptors (usually designated P2X or P2X_n) in that they are based on a different fundamental means of transmembrane signal transduction: P2Y receptors operate through a G protein-coupled system, while P2X receptors are ligand-gated 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. P2X, cDNA was cloned from the smooth muscle of the rat vas deferens (Valera et al. (1994) Nature 371 :516-519) and P2X₂ cDNA was cloned from PC 12 cells (Brake et al. (1994) Nature 371 :519-523). Five other P2X receptors have been found in rat cDNA libraries by virtue of their sequence similarity to P2Xι and P2X₂ (P2X₃: Lewis 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) ./. Neurosci. 16:448-455, Bo et al. (1995) FEBS Lett. 375: 129-133, Soto et al. (1996) Proc. Natl. Acad. Sci. USA 93:3684-3688, Wang et al. ( 1 96) Biochem. Biophys. Res. Commun.220: 196-202; P2X₅: Collo et al.

(1996) J. Neurosci.16:2495-2507, Garcia-Guzman 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: Surprenant et al. (1996) Science 272:735-738). For a comparison of the amino acid sequences of rat P2X receptors see Buell et al. (1996) Eur. J. Neurosci. 8:2221 -2228. Native P2X receptors form rapidly activated, nonselective cationic channels that are activated by ATP. Rat P2X, and rat P2X₂ have equal permeability to Na⁺ and K⁺ but significantly less to Cs . The channels formed by the P2X receptors generally have high Ca^" permeability (P_Ca/P_Na 4). The cloned rat P2X_j, P2X₂ and P2X₄ receptors exhibit the same permeability for Ca observed with native receptors. However, the mechanism by which P2X receptors form an ionic pore or bind ATP is not known-

A variety of tissues and cell types, including epithelial, immune, muscle and neuronal. express at least one form of P2X receptor. In rat, the distribution of the P2X3 receptor appears to be primarily in sensory ganglia like the dorsal root, trigeminal and nodese ganglia. However, study of the role of individual P2X receptors is hampered by the lack of receptor subtype- specific agonists and antagonists. For example, one agonist useful for studying ATP-gated channels is α,β-methylene-ATP (α,βmeATP). However, the P2X receptors display differential sensitivity to the agonist with P2Xι and P2X₂ being α,βmeATP-sensitive and insensitive, respectively. The predominant forms of P2X receptors in the rat brain, P2X₄ and P2X_ή receptors, cannot be blocked by suramin or PPADS. These two forms of the P2X receptor are also not activated by α,βmeATP and are, thus, intractable to study with currently available pharmacological tools.

A therapeutic role for P2 receptors has been suggested, for example, for cystic fibrosis (Boucher et αl. (1995) in: Belardinelli et αl. (eds) Adenosine αndAdenine Nucleotides: From Molecular Biology to Integrative Physiology (Kluwer Acad., Norwell MA) pp 525-532), diabetes (Loubatieres-Mariani et al. (1995) in: Belardinelli et al. (eds), supra, pp 337-345), immune and inflammatory diseases (Di Virgilio et al. (1 95) in: Belardinelli 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: Pathophysiology and Management in Adults and

Children (Wrightson Biomedical. Bristol) pp 41-49), behavioral disorders such as epilepsy, depression and aging-associated degenerative diseases (Williams (1993) Drug. Dev. Res. 28:438-444), contraception and sterility (Foresta et al. (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 P2 receptor-specific agonist and antagonist compounds.

- 2 - Summary 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 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 of producing recombinant P2X₃ polypeptides using such cells.

In yet another embodiment, the invention is directed to a method of expressing a human P2X₃ receptor, or a subunit thereof, in a cell to produce the resultant P2X₃- containing receptor.

In a further embodiment, the invention is directed to a method of 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 disclosure herein.

Brief Description of the Drawings

FIGURE 1 depicts the sequence of the P2X₃ 5' RACE product of Example 2 (SEQ ID NO: 13), in which the sequences of primers are underlined and the predicted initiation codon (ATG) is shown in boldface.

FIGURE 2 depicts the sequence of the P2X₃ 3 'RACE product of Example 3 (SEQ ID NO: 14), in which the sequences of primers are underlined and the predicted termination codon (TAG) is shown in boldface.

FIGURE 3 depicts the sequence of the complete open reading frame of cDNA encoding human P2X₃ receptor polypeptide (SEQ ID NO: 15). The initiation (ATG) and termination (TAG) codons are shown in boldface; 5' and 3' flanking sequences introduced during plasmid construction, including the EcoRI (GAATTC) and Not I (GCGGCCGC) restriction sites, are underlined.

3 - FIGURE 4 depicts the aligned predicted amino acid sequences of human (hP2X₃) (SEQ ID NO: 16) and rat (rP2X₃) (SEQ ID NO: 17) P2X₃ receptor polypeptides. Identical residues are identified by boxing.

Detailed Description of the Invention

The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology, microbiology, recombinant DNA technology, electrophysiology, and pharmacology, that are within the skill of the art. Such techniques are explained fully in the literature. See, for example, Sambrook, Fritsch & Maniatis, Molecular Cloning: A Laboratory Manual, Second Edition (1989); DNA Cloning, Vols. I and II (D.N. Glover ed. 1985); Perbal, B., A Practical Guide to Molecular Cloning (1984); the 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 (J. H. Miller et al. eds. ( 1987) Cold Spring Harbor Laboratory, Cold Spring Harbor, N. Y.); Scopes, Protein Purification: Principles and Practice (2nd ed., Springer- Verlag); and PCR: A Practical Approach (McPherson et al. eds. (1991) IRL Press).

All patents, patent applications and publications cited herein, whether supra or infra, are hereby incorporated by reference in their entirety and are deemed 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. Thus, for example, reference to "a primer" includes two or more such primers, reference to "an amino acid" includes more than one such 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" intends a purinergic receptor for the ligand ATP and/or other purine or pyrimidine nucleotides, whether natural or synthetic. P2 receptors are broadly subclassified as "P2X" or "P2Y" receptors. These types differ in their pharmacology, structure, and signal transduction mechanisms. The P2X receptors are generally ligand- gated ion channels, while the P2Y receptors operate generally through a G protein-coupled system. Moreover, and without intending to be limited by theory, it is believed that P2X receptors comprise multimers of receptor polypeptides, which multimers may be of either the same or different subtypes. Consequently, the term "P2X receptor" refers, as

- 4 - appropriate, to the individual receptor subunit or subunits, as well as to the homomeric and heteromeric receptors comprised thereby.

The term "P2X_n" intends a P2X receptor subtype wherein n is an integer of at least 1. At the time of the invention, at least 7 P2X_n receptor subtypes have been isolated and/or characterized.

A "P2X₃ receptor agonist" is a compound that binds to and activates a P2X₃ receptor. By "activates" is intended the elicitation 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-stranded DNA, as well as double- and single-stranded RNA. It also includes modifications, such as by methylation and/or by capping, and unmodified forms of the polynucleotide.

The term "variant" is used to refer to an oligonucleotide sequence which differs from the related wild-type sequence in 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, indicates a polypeptide sequence that differs from the wild-type polypeptide in the insertion, deletion 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 in function relative to the wild-type.

The term "mutant" generally refers to an organism or a cell displaying a new genetic character or phenotype as the result of change in its gene or chromosome. In some instances, however, "mutant" may be used in reference to a variant protein or oligonucleotide and "mutation" may refer to the change underlying the variant. "Polypeptide" and "protein" are used interchangeably herein and indicate a molecular chain of amino acids linked through peptide bonds. The terms do not refer to a specific length of the product. Thus, peptides, oligopeptides, and proteins are included within the definition 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 or variant proteins, fusion proteins and the

5 - like are included within the meaning of polypeptide, provided that such fragments, etc. retain the binding or other characteristics necessary for their intended use.

A "functionally conservative mutation" as used herein intends a change in a polynucleotide encoding a derivative polypeptide in which the activity is not substantially altered compared to that of the polypeptide from which the derivative is made. Such derivatives may have, for example, amino acid insertions, deletions, or substitutions in the relevant molecule that do not substantially affect its properties. For example, the derivative can include conservative amino acid substitutions, such as substitutions which preserve the general charge, hydrophobicity/hydrophilicity, side chain moiety, and/or steric bulk of the amino acid substituted, for example, Gly/Ala, Val Ile/Leu, Asp/Glu, Lys/Arg, Asn/Gln, Thr/Ser, and Phe/Trp Tyr.

By the term "structurally conservative mutant" is intended a polynucleotide containing changes in the nucleic acid sequence but encoding a polypeptide having the same amino acid sequence as the polypeptide encoded by the polynucleotide from which the degenerate variant is derived. This can occur because a specific amino acid may be encoded by more than one "codon," or sequence of three nucleotides, i.e., because of the degeneracy of the genetic code.

"Recombinant host cells," "host cells," "cells," "cell lines," "cell cultures," and other such terms denoting microorganisms or higher eukaryotic cell lines cultured as unicellular entities refer to cells which can be, or have been, used as recipients for recombinant vectors or other transfer DNA, immaterial of the method by which the DNA is introduced into the cell or the subsequent disposition 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 also can be used as recipients. A "vector" is a replicon in which another polynucleotide segment is attached, such as to bring about the replication and/or expression of the attached 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 start codon at the 5'-terminus and a translation stop codon at the 3'- terminus. A coding sequence can include, but is not limited to, mRNA, cDNA, and recombinant polynucleotide sequences. Variants or analogs may be prepared by the deletion of a portion of the coding sequence, by insertion of a sequence, and/or by substitution of one or more nucleotides within the sequence. Techniques for modifying nucleotide sequences, uch 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 wherein the components described are in a relationship permitting them to function in their intended manner. Thus, for example, a control sequence "operably linked" to a coding sequence is ligated in such a manner that expression of the coding sequence is achieved under conditions compatible with the control sequences. A coding sequence may 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, irrespective of the method used for the insertion, or the molecular form of the polynucleotide that is inserted. The insertion of a polynucleotide per se and the insertion of a plasmid or vector comprised of the exogenous polynucleotide are included. The exogenous polynucleotide may be directly transcribed and translated by the cell, maintained as a nonintegrated vector, for example, a plasmid, or alternatively, may be stably integrated into the host genome. "Transfection" generally is used in 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 also may refer to the formation of a cancerous or tumorigenic state.

The term "isolated," when referring to a polynucleotide or a polypeptide, intends that the indicated molecule is present in the substantial absence of other similar biological macromolecules. The term "isolated" as used herein means that at least 75 wt.%, more preferably at least 85 wt.%, more preferably still at least 95 wt.%, and most preferably at least 98 wt.% 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 may include functionally and/or structurally conservative mutations as defined herein.

A "test sample" as used herein intends a component of an individual's body which is a source of a P2X₃ receptor. These test samples include biological samples which can be evaluated by the methods of the present invention described herein and include body fluids such as whole blood, tissues and cell preparations. The following single-letter amino acid abbreviations are used throughout the text:

Alanine A Arginine R

Asparagine N Aspartic acid D

Cysteine C Glutamine Q

Glutamic acid E Glycine G

Histidine H Isoleucine I

Leucine L Lysine K

Methionine M Phenylalanine F

Proline P Serine S

Threonine T Tryptophan W

Tyrosine Y Valine V

A human P2X₃ receptor, a polynucleotide encoding the variant receptor or polypeptide subunits thereof, and methods of making the receptor are provided herein. The invention includes not only the P2X₃ receptor but also methods for screening compounds using the receptor and cells expressing the receptor. Further, polynucleotides and antibodies which can be used in methods for detection of the receptor, as well as the reagents useful in these methods, are provided. Compounds and polynucleotides useful in regulating the receptor and its expression also are provided as disclosed hereinbelow.

In one preferred embodiment, the polynucleotide encodes a human P2X₃ receptor polypeptide or a protein variant thereof containing conservative amino acid substitutions. DNA encoding the human P2X₃ receptor and variants thereof can be derived from 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 the preparation of

RNA using methods well known in the art (see, Sambrook et al., supra), or as a molecular probe capable of selectively hybridizing to, and therefore detecting the presence of, other

P2X₃-encoding nucleotides sequences. cDNA encoding the P2X₃ receptor may be obtained from an appropriate DNA library. cDNA libraries may be probed using the procedure described by Grunstein et al.

(1975) Proc. Natl. Acad. Sci. USA 73:3961. The cDNA thus obtained can then be modified and amplified using the polymerase chain reaction ("PCR") and primer sequences to obtain the DNA encoding the human P2X₃ receptor.

More particularly, PCR employs short oligonucleotide primers (generally 10-20 nucleotides in length) that match opposite ends of a desired sequence within the DNA

- 8 molecule. The sequence between the primers need not be known. The initial template can be either RNA or DNA. If RNA is used, it is first reverse transcribed to cDNA. The cDNA is then denatured, using well known techniques such as heat, and appropriate oligonucleotide primers are added in molar excess. Primer extension is effected using DNA polymerase in the presence of deoxynucleotide triphosphates or nucleotide analogs. The resulting product includes the respective primers at their 5'-termini, covalently linked to the newly synthesized complements of the original strands. The replicated molecule is again denatured, hybridized with primers, and so on, until the product is sufficiently amplified. Such PCR methods are described in for example, U.S. Patent Nos. 4,965,188; 4,800,159; 4,683,202; 4,683,195; incorporated herein by reference in their entireties. The product of the PCR is cloned and the clones containing the P2X₃ receptor DNA, derived by segregation of the primer extended strand, selected. Selection can be accomplished using a primer as a hybridization probe. Alternatively still, the P2X₃ receptor DNA could be generated using an RT-PCR

(reverse transcriptase - polymerase chain reaction) approach starting with human RNA. Human RNA may be obtained from cells or tissue in which the P2X₃ receptor is expressed, as for example dorsal root ganglion, trigeminal ganglion, pituitary gland, nodose ganglion or heart, using conventional methods. For example, single-stranded cDNA is synthesized from human RNA as the template using standard reverse transcriptase procedures and the cDNA is amplified using PCR. This is but one example of the generation of P2X₃ receptor variant from a human tissue RNA template.

Synthetic oligonucleotides may be prepared using an automated oligonucleotide synthesizer such as that described by Warner (1984) DNA 3:401. If desired, the synthetic τ strands may be labeled with "P by treatment with polynucleotide kinase in the presence of τ

"P-ATP, using standard conditions for the reaction. DNA sequences, including those isolated from genomic or cDNA libraries, may be modified by known methods which include site-directed mutagenesis as described by Zoller (1982) Nucleic Acids Res. 10:6487. Briefly, the DNA to be modified is packaged into phage as a single stranded sequence, and converted to a double stranded DNA with DNA polymerase using, as a primer, a synthetic oligonucleotide complementary to the portion of the DNA to be modified, and having the desired modification included in its own sequence. Culture of the transformed bacteria, which contain replications of each strand of the phage, are plated in agar to obtain plaques. Theoretically, 50% of the new plaques contain phage having the mutated sequence, and the remaining 50 have the original sequence. Replicates of the plaques are hybridized to labeled synthetic probe at temperatures and conditions suitable for hybridization with the correct strand, but not with the unmodified sequence. The sequences which 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 variant from wild-type by hybridization. In any case, the DNA would be sequence- confirmed.

Once produced, DNA encoding the P2X₃ receptor may then be incorporated into a cloning vector or an expression vector for replication in a suitable host cell. Vector construction employs methods known in the art. Generally, site-specific DNA cleavage is performed by treating with suitable restriction enzymes under conditions that generally are 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 cleaved fragments may be separated using, for example, polyacrylamide or agarose gel electrophoresis methods, according to methods known by those of skill in the art.

Sticky end cleavage fragments may be blunt ended using E. coli DNA polymerase 1 (Klenow) in the presence of the appropriate deoxynucleotide triphosphates (dNTPs) present in the mixture. Treatment with S 1 nuclease also may be used, resulting in the hydrolysis of any single stranded DNA portions. Ligations are performed using standard buffer and temperature conditions using T4

DNA ligase and ATP. Alternatively, restriction enzyme digestion of unwanted fragments can be used to prevent ligation.

Standard vector constructions generally include specific antibiotic resistance elements. Ligation mixtures are transformed into a suitable host, and successful transformants selected by antibiotic resistance or other markers. Plasmids from the transformants can then be prepared according to methods known to those in the art usually following a chloramphenicol amplification as reported by Clewell et al. (1972) J. Bacteriol. 1 10:667. The DNA is isolated and analyzed usually by restriction enzyme analysis and/or sequencing- Sequencing may be by the well-known dideoxy method of Sanger et al. (1977) Proc. Natl. Acad. Sci. USA 74:5463) as further described by Messing et al. (1981) Nucleic Acid Res. 9:309, or by the method reported by Maxam et al. (1980) Meth. Enzymol. 65:499. Problems with band compression, which are sometimes observed in GC rich regions, are overcome by use of, for example, T-deazoguanosine or inosine, according to the method reported by Barr et al. (1986) Biotechniques 4:428.

10 - Host cells are genetically engineered with the vectors of this invention, which may be a cloning vector or an expression vector. The vector may be in the form of a plasmid. a viral particle, a phage. etc. The engineered host cells can be cultured in conventional nutrient media modified as appropriate for activating promoters, selecting transformants/transfectants or amplifying the subunit-encoding polynucleotide. The culture conditions, such as temperature, pH and the like, generally are similar to those previously used with the host cell selected for expression, and will be apparent to those of skill in the art.

Both prokaryotic and eukaryotic host cells may be used for expression of desired coding sequences when appropriate control sequences that are compatible with the designated host are used. For example, among prokaryotic hosts, Escherichia coli is frequently used. Also, 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 that contains operons conferring ampicillin and tetracycline resistance, and the various pUC vectors, that also contain sequences conferring antibiotic resistance markers. These markers may 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 lambda- derived PI promoter and N gene ribosome binding site (Shimatake et α/. (1981) Nature 292: 128), the hybrid Tac promoter (De Boer et al. (1 83) Proc. Natl. Acad. Sci. USA 292: 128) derived from sequences of the trp and lac UV5 promoters. The foregoing systems are particularly compatible with E. coli: however, other prokaryotic hosts such as strains of Bacillus or Pseudomonas may be used if desired.

Eukaryotic hosts include yeast and mammalian cells in culture systems. Pichia pastoris, Saccharomyces cerevisiae and S. carlsbergensis are commonly used yeast hosts. Yeast-compatible vectors carry markers that permit selection of successful transformants by conferring protrophy to auxotrophic mutants or resistance to heavy metals on wild-type strains- Yeast-compatible vectors may employ the 2-μ origin of replication (Broach et al. ( 1983) Metb. Enzymol. 101 :307), the combination of CEN3 and ARS 1 or other means for assuring replication, such as sequences that will result in incorporation of an appropriate fragment into the host cell genome. 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) ./. Adv. Enzyme Reg. 7: 149, Holland et al. ( 1978) Biochemistry 17:4900 and Hkzeman ( 1 80) ./. Biol. Chem. 255:2073. For example, some useful control systems are those that comprise the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) promoter or alcohol dehydrogenase ( ADH) regulatable promoter, or the hybrid yeast promoter ADH2/GAPDH described in Cousens et al. Gene ( 1987) 61 :265-275, terminators also derived from

GAPDH, and, if secretion is desired, leader sequences from yeast alpha factor. In addition, the transcriptional regulatory region and the transcriptional initiation region which are operably linked may be such that they are not naturally associated in the wild-type organism. Mammalian cell lines available as hosts for expression are known in the art 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, baby hamster kidney (BHK) cells, and others. Suitable promoters for mammalian cells also are known in the art and include viral promoters such as that from Simian Virus 40 (SV40), Rous sarcoma virus (RSV), adenovirus (ADV), bovine papilloma virus (BPV) and cytomegalovirus (CMV). Mammalian cells also may require terminator sequences and poly A addition sequences; enhancer sequences which increase expression also may be included, and sequences which cause amplification of the gene also may be desirable. These sequences are known in the art. Vectors suitable for replication in mammalian cells may include viral replicons, or sequences which ensure integration of the appropriate sequences encoding the P2X₃ receptor into the host genome. An example of such a mammalian expression system is described in Gopalakrishnan et al. (1995), Eur. ./.

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 Stϋhmer ( 1992) Meth.

Enzymol. 207:319-345, insect cells using 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 for high level of protein expression, while post-translationally processing the protein in a manner similar to mammalian cells. These expression systems use viral promoters that are activated following baculovirus infection to drive expression of cloned genes in the insect cells (O'Reilly et al.

(1992) Baculovirus Expression Vectors: A Laboratory Manual, IRL/Oxford University

Press). Transfection may be by any known method for introducing polynucleotides into a host cell, including packaging the polynucleotide in a virus and transducing a host cell with the virus, by direct uptake of the polynucleotide by the host cell, and the like, which methods are known to those skilled in the art. The transfection procedures selected depend upon the host to be transfected and are determined by the rountineer.

The expression of the receptor may be detected by use of a radioligand selective for the receptor. However, any radioligand binding technique known in the art may be used to detect the receptor (see, for example, Winzor et al. (1995) Quantitative Characterization of Ligand Binding, Wiley-Liss, Inc., NY; Michel et al. ( 1997) Mol. Pharmacol. 51 :524- 532). Alternatively, expression can be detected by utilizing antibodies or functional measurements, i.e., ATP-stimulated cellular depolarization using methods that are well known to those skilled in the art. For example, agonist-stimulated Ca influx, or inhibition by antagonists of agonist-stimulated Ca influx, can be measured in mammalian cells transfected with the recombinant P2X₃ receptor cDNA, such as COS, CHO or HEK cells. Alternatively, Ca influx can be measured in cells that do not naturally express P2 receptors, for example, the 1321N1 human astrocytoma 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 recombinant host cell cultures expressing the same by known methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, hydroxyapatite chromatography or lectin chromatography- Protein refolding steps can be used, as necessary, in completing configuration of the protein. Finally, high performance liquid chromatography (HPLC) can be employed for final purification steps. The human P2X₃ receptor polypeptide, or fragments thereof, of the present invention also may 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 either solid or solution phase synthesis methods. See, for example, J. M. Stewart and J. D. Young, Solid 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, Principles of Peptide Synthesis, Springer- Verlag, Berlin ( 1984) and E. Gross and J. Meienhofer, Eds., The Peptides: Analysis, Synthesis. Biology, supra. Vol. 1 , for classical solution synthesis.

13 In one preferred system, either the DNA or the RNA derived therefrom, each of which encode the human P2X₃ receptor, may 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 the mRNA can be evaluated as follows. A receptor- encoding polynucleotide is injected into an oocyte for translation into a functional receptor subunit- The function of the expressed variant human P2X₃ receptor can be assessed in the oocyte by a variety of techniques including electrophysiological techniques such as voltage- clamping, and the like.

Receptors expressed in a recombinant host cell may be used to identify compounds that modulate P2X₃ activity. 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 from these cells. This may be done by measuring specific binding of labeled (for example, radioactive) compound to the cells, cell membranes or isolated receptor, or by measuring the ability of the compound to displace the specific binding of a standard labeled ligand. See, Michel et al., supra. Expression of variant receptors and screening for compounds that bind to, or inhibit the binding of labeled ligand to these cells or membranes, provide a method for rapid selection of compounds with high affinity for the receptor. These compounds may be agonists, antagonists or modulators of the receptor. Expressed receptors also may be used to screen for compounds that modulate P2X₃ receptor activity. One method for identifying compounds that modulate P2X₃ activity, comprises providing a cell that expresses a human P2X₃ receptor polypeptide, combining a test compound with the cell and measuring the effect of the test compound on the P2X₃ receptor activity- The cell may be a bacterial cell, a mammalian cell, a yeast cell, an amphibian cell, an insect or any other cell expressing 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 several disorders including, without limitation, central nervous system or peripheral nervous system conditions, for example, epilepsy, pain, depression, neurodegenerative diseases, and the like, and in disorders of the reproductive system, asthma, peripheral vascular disease, hypertension, immune system disorders, 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)

14 Nature 477:385-386: Chen et al. ( 1995), supra). Consequently, it is believed that the screening methods of the present invention may be especially suitable for the identification of compounds useful as analgesic and anti-nociceptive agents.

In addition, the 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 comprised of a polynucleotide, as defined above, which contains a nucleic acid sequence complementary to a nucleic acid sequence present in a target 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 from wild-type message, with the proviso that it may be difficult to design a method capable of making such a distinction given the small differences that may exist between sequences coding the wild-type and a variant P2X receptor. Alternatively, a PCR-based assay could be used to amplify the sample RNA or DNA for sequence analysis. Furthermore, the P2X₃ polypeptide or fragment(s) thereof can be used to prepare monoclonal antibodies using techniques that are well known in the art. The P2X₃ receptor or relevant fragments can be obtained using the recombinant technology outlined below, i.e., a recombinant cell that expresses the receptor or fragments can be cultured to produce quantities of the receptor or fragment that can be recovered and isolated. Alternatively, the P2X₃ polypeptide or fragment(s) thereof can be synthesized using conventional polypeptide synthetic techniques as known in the art. Monoclonal antibodies that display specificity and selectivity for the P2X₃ polypeptide can be labeled with a measurable and detectable moiety, for example, a fluorescent moiety, radiolabels, enzymes, chemiluminescent labels and the like, and used in in vitro assays. It is theorized that such antibodies could be used to identify wild-type or variant P2X₃ receptor polypeptides for immunodiagnostic purposes.

For example, antibodies have been generated to detect amyloid bl-40 v. 1-42 in brain tissue (Wisniewski et al. (1996) Biochem. J. 313:575-580; also see, Suzuki et al. (1994) Science 264: 1336-1340; Gravina et α/. (1995) J. Blol. Chem. 270:7013- 7016; and Turnet et al. (1996) J. Blol. 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.

15 - Example 1 Identification of a Human cDNA Sequence Likely to Encode P2X. Polypeptide

The predicted amino acid sequence of the rat P2X₃ receptor (NCBI sequence I.D. number 1 103623) was used to search for human DNA sequences which would code for similar polypeptides. The TBLASTN database search tool (Altschul (1993) J. Mol. Evol. 36:290-300) was used, which allows querying nucleotide databases with a protein sequence by dynamically translating the DNA sequences into all 6 possible reading frames. A search of the Genbank sequence-tagged sites (STS) database revealed a human genomic fragment, 229 basepairs in length, containing an open reading frame which would be predicted to encode a polypeptide having a high degree of homology to a region of the rat P2X₃ receptor. The deposited sequence for this fragment (Genbank accession number G03901 ) was as follows:

CCCGAATCGG TGGACTGCTT CTCCACTGTG GTCTGGTCGC TGGGGTACAC TGGGTTGGTC AAAGCCGCGA TTTTCAGTGT AGTCTCATTC ACNTGNAGGC GAAAGAGCTG GTGTTGTCAA GTTCTGACTA TGGGCAATGT CCTCTTTTGT GACCCCATTT GACAGACTCA GCAGTGGGCG CCCATGACCT AGTCATGAGG GGAGCCAGGA CATCTGTGTG ATCCCAAGG (SEQ ID NO : 1 )

where "N" represents any of the bases A, T, G and C.

Example 2 Identification of the 5' End of the P2X₂ cDNA

Based on the sequence of G03901, primers were designed for use in reverse- transcription polymerase chain reaction (RT-PCR) procedures in an effort to isolate the intact open reading frame for this receptor. The primers used in the reactions described below were as follows:

16 - Primer I s (SEQ ID. NO:2):

5'-TTTACCAACCCAGTGTACCC-3'

Primer 2s (SEQ ID. NO:3):

5 -ACCACAGTGGAGAAGCAGTC-3'

Primer 3as (SEQ ID. NO:4):

5'-GAATCGGTGGACTGCTTCTC-3'

Primer 4as (SEQ ID. NO:5):

5'-CGATTTTCAGTGTAGTCTCATTC-3^,

Primer 5as (SEQ ID. NO:6): 5 -GGGGTACACTGGGTTGGTAA-3'

5' RACE Anchor Primer (SEQ ID. NO:7):

5'-CUACUACUACUAGGCCACGCGTCGACTAGTACGGGIIGGGIIGGGIIG-

3' (where "U" represents uracil and "I" represents inosine)

Universal Adapter Primer (SEQ ID. NO:8):

5'-CUACUACUACUAGGCCACGCGTCGACTAGTAC-3'

Adapter Primer (SEQ ID. NO:9):

5'-GGCCACGCGTCGACTAGTACTTTTTTTTTTTTTTTTT-3'

Abridged Universal Adapter Primer (SEQ ID. NO: 10): 5'-GGCCACGCGTCGACTAGTAC-3'

5'hP2X₃ Primer (SEQ ID. NO: l 1):

5'-CACCATGAACTGCATATCCGACTTC-3' 3'hP2X₃ Primer (SEQ ID. NO: 12):

5'-CTAGTGGCCTATGGAGAAGGC-3'

To identify the 5' end of the cDNA which is derived from the genomic region which sequence G03901 is pan of, the RACE technique (Rapid Amplification of cDNA Ends) (Frohman et al. ( 1 88), Proc. Natl. Acad. Sci. U.S. A. 85:8998-9002) was employed. Extension of the cDNA identified through the RT-PCR step was accomplished using the 5'RACE™ reagent system (Life Technologies, Gaithersburg, MD). One microgram of poly A+ RNA derived from human pituitary gland tissue (Cat. # 65894-1, Lot # 6080167; Clontech Laboratories. Palo Alto, CA) was used in a reaction using reagents provided in the kit as described; l μl (1 μg) of RNA was combined with 3 μl (3pmoI) Primer 3as and 1 1 μl

RNase-free water (water treated with diethylpyrocarbonate, or DEPC) and heated to 70"C for 10 minutes followed by 1 minute on ice. 2.5 μl l()x reaction buffer (200 mM Tris-HCl pH 8.4, 500mM KC1). 3 μl 25 mM MgCl₂, 1 μl 10 mM dNTP mix. and 2.5 μl 0.1 M DTT were added. The mix was incubated at 42°C for 2 minutes after which 1 μl Superscript II™ 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 kit. The cDNA was eluted from the column in 50 μl distilled water (dH_^O), lyophilized, and resuspended in 21 μl dH₂O. Tailing of the cDNA was accomplished in the following reaction: 7.5 μl dH_:O. 2.5 μl reaction buffer (200 mM Tris-HCl pH 8.4, 500mM KC1), 1.5 μl 25 mM MgCl,, 2.5 μl 2mM 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.

PCR amplification of the cDNA was performed in the following steps: 5 μl of the cDNA was included in a reaction which also contained 5 μl lOx Gene Amp™ PCR buffer

(Perkin Elmer, Foster City, CA) (500mM KC1, lOOmM Tris-HCl pH 8.3, 15mM MgCL. and 0.0 l%(w/v) gelatin), 1 μl 10 mM dNTP mix. lμl ( 10 pmol) Anchor Primer, 1 μl ( 10

18 - pmol) Primer 5as. and 35 μl dH,O. The reaction was heated to 95°C for 1 minute, then held at 80°C for 2 minutes, during which 0.5 μl (2.5 units) Amplitaq™ polymerase (Perkin-

Elmer) was added. The reaction was cycled 35 times under these conditions: 94°C for 15 seconds. 52°C for 20 seconds, and 72"C for 1 minute. After the amplification, the reaction products were purified utilizing the QiaQuick™

PCR product purification system (Qiagen. Inc., Chatsworth CA) as per the manufacturer's instructions- The products were eluted from the columns with 50 μl TE buffer (lOmM Tris, lm-M EDTA pH 8.0), and one microliter of the eluent was utilized as template DNA in a PCR reaction to increase levels of specific product for subsequent isolation. The reamplification also included: 5 μl lOx GeneAmp™ PCR buffer, 1 μl 10 mM dNTP mix, 1 μl (10 pmol) Universal Amplification Primer, 1 μl (10 pmol) Primer 4as, and 40.5 μl dH₂O. The reaction was heated to 95°C for 1 minute, then held at 80°C during which 0.5 μl

(2.5 units) Amplitaq™ polymerase was added. The reaction was cycled 35 times under these conditions; 94°C for 15 seconds, 50"C for 20 seconds, and 72°C for 1 minute. Amplification products were analyzed via 0.87c agarose gel electrophoresis and a predominant product of approximately 1.3 kilobase pairs in length was detected. This product was excised from the gel and purified via the QiaQuick™ purification system. The product was eluted from the column with 50 μl dH₂0 and lyophilized to 10 μl volume.

Three microliters of the resulting DNA was used in a ligation reaction with pCR 2.1 vector (Invitrogen, Carlsbad, CA) incubated at 14°C overnight. The ligation products were used to transform E. coli from the cloning kit using standard manufacturer's protocols. Insert sizes of 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™, Perkin Elmer Applied Biosystems Division, Foster City, CA) and an Applied Biosystems Model 373 DNA sequencer. The sequence of the 5'RACE product including the EcoRI sites from the pCR 2.1 vector is shown in Figure 1 (SEQ ID NO: 13). The sequences of the amplimers (Universal Amplification Primer and the complement to Primer 4as) are underlined.

19 - Example 3 Identification of the 3' End of the P2X, cDNA

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 employed with primers designed to STS G03901. Poly A+ RNA (500 nanograms) derived from pituitary gland tissue (see Example 2, above) was used in the reaction as follows: The RNA and 10 picomoles Adapter Primer were combined in a final volume of 12 μl dH₂O. This mixture was heated to 70°C for 10 minutes and chilled on ice for 1 minute. The following components were added: 2 μl lOx PCR buffer (200 mM Tris-HCl pH 8.4, 500mM KC1), 2 μl 25 mM MgCl₂, 1 μl lOmM dNTP mix, and 2 μl 0.1M dithiothreitol. The reaction was equilibrated to 42°C for 2 minutes after which 1 μl (200 units) Superscript IJ™ reverse transcriptase was added and incubation continued at 42°C for 50 minutes. The reaction was terminated by incubation at 70°C for 15 minutes and chilled on ice. Rnase H (1 μl; 2 units) was added and the mixture was incubated for 20 minutes at 37°C, then stored on ice.

Amplification of the 3' end of the P2X₃ cDNA was accomplished in the following reactions: 2μl of the first strand cDNA synthesized above was used in a PCR mixture also including 5 μl lOx Gene Amp™ PCR buffer, 1 μl lOmM dNTPs, 1 μl (10 picomoles) Primer Is, lμl (10 picomoles) Abridged Universal Amplification Primer (AUAP) and 39.5 μl dH₂O. The reaction was heated to 95°C for 1 minute, then held at 80°C for 2 minutes, during which 0.5 μl (2.5 units) Amplitaq™ polymerase was added. The reaction was cycled 35 times under these conditions; 94°C for 15 seconds, 54°C for 20 seconds, and 72°C for 2minutes. After cycling, the reaction was incubated for 10 minutes at 70°C and stored at 4°C.

After the amplification, the reaction products were purified utilizing the QiaQuick™ PCR product purification system as per the manufacturer's instructions. The products were eluted from the columns with 50 μl TE buffer (lOmM Tris, 0.1 mM EDTA pH 8.0) and one microliter of the eluent was utilized as template DNA in a PCR reaction to increase levels of specific product for subsequent isolation. The reamplification also included: 5 μl lOx GeneAmp™ PCR buffer, 1 μl 10 mM dNTP mix, 1 μl ( 10 pmol) AUAP, 1 μl (10 pmol) Primer 2s, and 40.5 μl dH,O. The reaction was heated to 95°C for 1 minute, then held at

20 - 80°C during which 0.5 μl (2.5 units) Amplitaq™ polymerase was added. The reaction was cycled 35 times under these conditions; 94°C for 15 seconds, 54°C for 20 seconds, and 72°C for 2 minutes. 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 excised from the gel and purified via the Qiaquick™ purification system. The product was eluted from the column with 50 μl dH₂0 and lyophilized to 10 μl volume.

Three 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 kit. Insert sizes of resulting clones were determined using ΕcoRI 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 an Applied Biosystems 373 DNA sequencer. The sequence of the 3'RACΕ product including the ΕcoRI sites from the pCR 2.1 vector is shown in Figure 2 (SΕQ ID NO: 14), in which the sequences of the amplimers (AUAP and the complement to Primer 2s) are underlined.

Example 4 Isolation of cDNA Containing the Intact Open Reading Frame of Human P2X₃

Using information on the sequence surrounding the initiation and termination codons of the human P2X₃ message, oligonucleotide primers were designed and synthesized to enable RT-PCR of the intact open reading frame of the mRNA. The sequences of these primers, designated 5'hP2X₃ and 3'hP2X₃, are shown above. PCR amplification was performed on a portion (2ul) of the pituitary gland cDNA described in Example 3. A proofreading thermostable polymerase (Cloned Pfu DNA Polymerase, Strategene, La Jolla, CA) was used in the amplification to ensure high-fidelity amplification. The reaction mixture consisted of 2 μl cDNA, 5 μl lOx cloned Pfu polymerase reaction buffer (200 mM Tris-HCl (pH 8.8), lOOmM KCl, lOOmM (NH )₂SO₄, 20mM MgSO , 1 % Triton X-100, 1 mg/ml nuclease-free bovine serum albumin), 1 μl dNTP mix, lμl (10 picomoles) 5'hP2X₃ Primer, lμl (10 picomoles) 3'hP2X₃ Primer, and 39.5 μl dH₂O. The reaction was heated to 95°C for 1 minute, then held at 80"C for 2 minutes, during which time 0.5 μl (1.25units) cloned Pfu polymerase was added. The reaction was cycled 35 times under the following conditions: 94°C for 20 seconds. 52°C for 20 seconds, and 72°C for 3.5 minutes. After cycling, the reaction was incubated for 10 minutes at 70°C. The reaction products were separated on a 0.8 7c agarose gel and a product of approximately 1.2 kilobases was excised and purified via the QiaQuick™ gel purification system. The DNA was eluted with 50 μl dH₂O, lyophilized and resuspended in 10 μl dH₂O- One microliter of this DNA was use in a reamplification reaction which also inluded

5 μl lOx Pfu reaction buffer, 1 μl dNTP mix, lμl (10 picomoles) 5'hP2X₃ Primer, l μl (10 picomoles) 3'hP2X₃ Primer, and 40.5 μl dH₂O. The reaction was heated to 95°C for 1 minute, then held at 80°C for 2 minutes, during which 0.5 μl (1.25units) cloned Pfu polymerase was added. The reaction was cycled 15 times under the following conditions: 94°C for 20 seconds, 52°C for 20 seconds, and 72°C for 3.5 minutes. After cycling, the reaction was incubated for 10 minutes at 70°C. The reaction products were separated on a 0.8 % agarose gel and the 1.2 kilobase product was excised and purified via the QiaQuick™ gel purification system. The DNA was eluted with 50 μl dH₂O, lyophilized and resuspended in 15 μl dH₂O. Three microliters of the purified PCR product was used in a ligation reaction using the pCRscript™ cloning system (Stratagene) which also included 0.5μl (5ng) of the pCRScript™ Amp SK(+) vector, l μl of pCRScript™ lOx Reaction Buffer, 0.5 μl of lOmM ATP, lμl (5 units) Srf I restriction enzyme, 1 μl (4 units) T4 DNA ligase, and 3 μl dH₂O. The reaction mixture was incubated at room temperature for one hour, then at 65°C for 10 minutes.

One microliter of this reaction product was used to transform XL-2 blue ultracompetent cells (Stratagene) as per standard manufacturer's protocols. Resulting clones were screened by restriction analysis and sequenced using fluorescent dye-terminator reagents (Prism, Applied Biosystems) and an Applied Biosystems Model 310 DNA sequencer. The sequence of the intact open reading frame is shown in Figure 3 (SEQ ID

NO: 15). A comparison of the predicted protein sequence of the human P2X₃ of the present invention (SEQ ID NO: 16) with that of the corresponding rat polypeptide (SEQ ID NO: 17) is depicted in FIGURE 4. Example 5 Expression and Electrophvsiological Analysis of Recombinant P2X_? Receptors in Xenopus Oocytes

Oocytes of Xenopus laevis were prepared and injected with receptor DNA of the present invention, and receptor responses were measured using two-electrode voltage- clamp, according to procedures previously described (Briggs et al. (1995), supra). Oocytes were maintained at 17-18°C in normal Barth's solution (90 mM NaCl, 1 mM KCl, 0.66 mM NaNO₃, 0.74 mM CaCl₂, 0.82 mM MgCl₂. 2.4 mM NaHCO₃, 2.5 mM sodium pyruvate, and 10 mM Na N-(2-hydroxy-ethyl)-piperazine-N'-(2-ethanesulfonic acid) ("HEPES") buffer, final pH 7.55) containing 100 μg/ml gentamicin. Responses were measured at a holding potential of -60 mV in modified Barth's solution containing 10 mM

BaCL and lacking CaCl₂ and MgCl₂ (final pH 7.4). However, in some experiments, the cell potential was intentionally varied in order to determine the response current-voltage relationship. Agonist was applied briefly using a computer-controlled solenoid valve and a push/pull applicator positioned to within 200-400 μm from the oocyte. Responses were recorded by computer in synchrony with agonist application. Antagonists were included with agonist in the push/pull applicator and were applied to the bath by superfusion for at least 3 minutes before application of agonist. Responses were quantified by measuring the peak amplitude.

DNA for injection into oocytes was the P2X₃ insert from pCDNA3.1 prepared as described in Example 2. The clone was grown up and prepared in large scale using the QIAgen maxiprep DNA preparation system according to the manufacturer's instructions- The DNA was ethanol precipitated and resuspended in TE buffer. For RNA production, the P2X₃-pCDN A3.1 construct was linearized by digestion with the restriction enzyme NotI and P2X₃ messenger RNA was produced using the T7 promoter in this vector and the

Ambion mMessage mMachine™ in vitro transcription kit according to the manufacturer's instructions.

For functional anaysis of human P2X, receptors, 10 ng of human P2X₃ DNA prepared as described above were injected into the nucleus of Xenopus oocytes. Oocytes were incubated in normal Barth's solution containing 100 μg/ml gentamicin for 2-7 days following injection. The response to 10 μM ATP was then recorded.

The results of the above expression and analysis show the receptors of the present invention to be functional. Oocytes injected with human P2X₃ DNA responded to

- 23 - extracellular application of ATP by 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 EC₅₀ of 0.7 μM was obtained from concentration-response relationships

(0.01- 1 00 μM) from these oocytes. ATP-induced current-voltage relationships were also recorded from these oocytes. These revealed a reversal potential of approximately zero mV, with pronounced inward rectification recorded at negative membrane potentials.

Another P2X receptor agonist, α,β-methylene-ATP, elicited maximal currents similar to those evoked by ATP, although it was slightly less potent (EC₅₀ = 2.1 μM). Application of a third P2X receptor agonist, 2-methylthio-ATP, was slighly more potent (EC₅₀ = 0.4 μM) than either ATP or cc,βmethylene-ATP. Functional antagonism of responses was determined by application of the non-specific P2X receptor antagonists suramin or pyridoxal-phosphate-6-azophenyl-2',4'-disulfonic acid (PPADS). Both antagonists produced a complete block of ATP (0.3 μM)-induced currents, with suramin displaying increased potency (IC₅₀ = 0.3 μM) relative to PPADS (IC₅₀ = 1 μM). In summary, injection of human P2X₃ receptor DNA into Xenopus oocytes resulted in expression of functional P2X₃ receptors on the cell surface, and these receptors function as ligand-gated non-specific cation channels. These receptors responded to extracellular P2 receptor agonists with a rank order potency of 2-methylthio-ATP > ATP > α,β-methylene-

ATP. They also exhibit inward rectification and are blocked by both P2 receptor antagonists PPADS and suramin.

Claims

What is claimed is:

1 . An isolated polynucleotide encoding a human P2X₃ receptor polypeptide or a degenerate variant thereof.

2. A polynucleotide according to Claim 1, wherein the polynucleotide is a polydeoxyribonucleotide (DNA).

3. A polynucleotide according to Claim 1, wherein the polynucleotide is a polyribonucleotide (RNA).

4. A polynucleotide according to Claim 2, wherein the DNA comprises the sequence of SEQ ID NO: 15.

5. A host cell comprising a polynucleotide according to Claim 1 or Claim 4.

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. A host cell according to Claim 6, wherein the cell is an amphibian cell.

8. A host cell according to Claim 6, wherein the cell is a mammalian cell.

9. An expression vector comprising a polynucleotide according to Claim 1 operably linked to control sequences that direct the transcription of the polynucleotide, whereby the polynucleotide is expressed in a host cell.

10. An expression vector according to Claim 9, wherein the human P2X₃ receptor polypeptide comprises the amino acid sequence of SEQ ID NO: 16.

1 1. A host cell comprising an expression vector according to Claim 9.

12. A host cell according to Claim 1 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. A host cell according to Claim 12, wherein the cell is an amphibian cell.

14. A host cell according to Claim 12. wherein the cell is a mammalian cell.

15. A host cell comprising the expression vector of Claim 10.

16. A host cell according to Claim 15, wherein the cell is selected from the group consisting of a bacterial cell, a mammalian cell, a yeast cell and an amphibian cell.

17. A host cell according to Claim 16, wherein the cell is an amphibian cell.

18. A host cell according to Claim 16, wherein the cell is a mammalian cell.

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 1 under conditions that allow the production of the polypeptide; and

(b) recovering the polypeptide.

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 the production of the polypeptide; and

(b) recovering the polypeptide.

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. 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 cell expressing the P2X receptor, or

(ii) the binding of the test compound to the cell or the P2X receptor.

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. A method according to Claim 22, wherein said measurement of step (c) (ii) is performed by measuring a signal generated by a detectable moiety.

25. A method according to Claim 24, wherein said detectable moiety is selected from the group consisting of a fluorescent label, a radiolabel, a chemiluminescent label and an enzyme.

26. A method according to Claim 22, wherein said measurement of step (c) (i) is performed by measuring a signal generated by a radiolabeled ion, a chromogenic reagent, a fluorescent probe or an electrical current.

27. A method according to Claim 23, wherein the host cell is a mammalian cell.

28. A method according to Claim 23, wherein the host cell is an amphibian cell.

29. A method according to Claim 22, wherein the human P2X₃ receptor polypeptide comprises the amino acid sequence of SEQ ID NO: 16.

30. A method for detecting a target polynucleotide of a P2X₃ receptor in a test sample, the method comprising the steps of:

(a) contacting the target polynucleotide with at least one human P2X₃ receptor-specific 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. A method for detecting cDNA of human P2X receptor mRNA 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. A method according to Claim 31 , wherein said detection step (c) comprises utilizing a detectable moiety capable of generating a measurable signal.

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 thereof.

34. A purified polynucleotide according to Claim 33, wherein the polynucleotide is produced by recombinant techniques.

35. A polypeptide encoded by human P2X₃ receptor polynucleotide wherein said polypeptide comprises the amino acid sequence of SEQ ID NO: 16 or a portion thereof.

36. A polypeptide according to Claim 35 produced by recombinant techniques.

37. A polypeptide according to Claim 35 produced by synthetic techniques.

38. A monoclonal antibody which specifically binds to human P2X₃ receptor comprising the amino acid sequence of SEQ ID NO: 16 or an immunoreactive fragment thereof.

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 which specifically binds to the human P2X₃ receptor, for a time and under conditions sufficient for the formation of a resultant complex; and

(b) detecting the resultant complex containing the antibody, wherein said antibody specifically binds to human P2X₃ receptor amino acid comprising the amino acid sequence of SEQ ID NO: 16 or a fragment thereof.

- 28 -