WO2003091281A1 - Pain receptor gene and protein - Google Patents

Abstract

The present invention is directed to a novel pain receptor protein,the nucleic acids encoding same andto vectors containing said nucleic acids as well as hosts transformed with said vectors; it also encompasses a transgenic non-human animal containing an isolated nucleic acid encoding said pain receptor, an antibody or aptamer directed against said pain receptor and a hybridoma, which produces monoclonal antibodies against said pain receptor. The invention comprises inhibitors and agonists of said pain receptors and compositions containing same as well as compositions containing said nucleic acids, proteins or antibodies/aptamers specific thereto. Furthermore, the present invention is directed to methods of making said proteins, methods of detecting the expression level of said protein, methods of detecting mutations in one of said nucleic acids and proteins, methods of pain inhibition as well as methods of screening for agonists/antagonists of said pain receptor proteins.

Description

PAIN RECEPTOR GENE AND PROTEIN

The present invention is directed to a novel pain receptor protein and the nucleic acids encoding same. The invention further relates to vectors containing said nucleic acids as well as hosts transformed with said vectors. The invention also encompasses a transgenic non-human animal containing an isolated nucleic acid encoding said pain receptor, an antibody or aptamer directed against said pain receptor as well as a hybridoma, which produces monoclonal antibodies against said pain receptor. According to a further aspect, the invention comprises inhibitors and agonists of said pain receptors and compositions containing same as well as compositions containing said nucleic acids, proteins or antibodies/aptamers specific thereto. Furthermore, the present invention is directed to methods of making said proteins, methods of detecting the expression level of said protein, methods of detecting mutations in one of said nucleic acids and proteins, methods of pain inhibition as well as methods of screening for agonists/antagonists of said pain receptor proteins.

The detection of pain by specific neurons within the peripheral nervous system is based on the ability of these cells to sense noxious thermal, chemical and/or mechanical stimuli which may be injurious for the individual. These nociceptors are equipped with a highly specific setup of proteins enabling the detection of those stimuli. During the last years, the identity of a growing number of "pain genes" has been elucidated^, especially a number of different ion channel genes involved in nociception have been cloned and charcaterized^.

Among them is the family of vanilloid receptors which currently consists of three members. The first one, NR1 (for vanilloid receptor 1), has been cloned in 1997 (ref. 3) due to its ability to respond to capsaicin (because the action of capsaicin depends on a vanilloid moiety within its chemical structure the gene was called "vanilloid receptor" and not "capsaicin receptor"). Capsaicin - the compound in peppers that makes them taste "hot" - is known to activate cation channels on nociceptors which leads to the perception of burning pain and a local inflammatory response. A prolonged application of capsaicin on the other hand is analgesic because of a desensitization of the nociceptor terminals which (in terms of a therapeutic potential) is one of the reasons why the understanding of the molecular basis of capsaicin action is one of the major issues in pain research. NRl is a non-selective cation channel with homology to the TRP family of capacitative Ca²⁺ channels which is activated by capsaicin. Moreover, it is also activated by increases in temperature in a potentially noxious range (>42°C) and by low pH. The analysis of NRl knockout mice confirms the involvement of this channel in pain sensation, as well as in hypersensitivity to noxious stimuli following tissue injury^. On the other hand, these data show the existence of redundant mechanisms for the sensation of heat-evoked pain.

Indeed, the second member of this family, NRL1 (for vanilloid receptor like 1 gene), can also be activated by heat, yet, temperatures over 52°C are needed^. On the other hand, NRL1 is not activated by vanilloids or protons.

Very recently, another homolog was cloned by two independent groups which named the channel NR-OAC (for vanilloid receptor related osmotically activated channel gene) and OTRPC4 (due to its homology to the TRP channels)^. This novel homolog also codes for a non-selective anion channel which in contrast to the other members is activated by hypotonicity and is localized in neurosensory cells responsive to systemic osmotic pressure. VR-OAC is not sensitive to vanilloids or heat up to 55°C. Just NRl is activated by vanilloids, yet, a number of different pharmacological studies postulate the existence of additional vanilloid receptors (see e. g. ref. 9).

The research already provided some molecular pieces in the puzzle of pain sensation. While much progress has been made in understanding the senses of taste and smell and sight at the molecular level, the molecules that underlie the perception of pain are just now being studied.

Certain successes have been achieved based on known pain receptors: genetic engineering allowed to take a closer look at what happens in whole animals when, for example, NRl cannot function¹⁰. Such gene 'knockout' mice encouragingly could better tolerate painful heat and hot food, however, pain was not cut off completely.

Therefore, there is still a need for novel and improved pain therapies based on the knowledge of novel families of pain receptors. Thus, the search for hitherto unknown homologs of the NR family could be a valuable tool to identify new pain receptors, which open up new approaches in the treatment of pain conditions of different origin.

Therefore it is an object of the present invention to provide a novel group of pain receptors. It is a further object of the present invention to provide substances, which are capable of an interaction with said novel pain receptors or are able to influence their expression.

These objects are solved by the subject-matters of the independent claims. Preferred embodiments are set forth in the dependent claims.

According to the present invention, a novel group of pain receptors is provided called NRL3. This group could be classified as the fourth member of the family of vanilloid receptors. NRL3 shows an overall homology to NRl, VRL1 and NR-OAC of about 62%, 56% and 62%, respectively, showing on the one hand a certain relationship between the respective pain receptors, but on the other hand also substantial differences in their sequences.

Further comparison with the other NR members showed that all four NRs are nearly equally related to each other and that there do not seem to be distinct subbranches within the NR family (see also figure 3).

According to the invention a pain receptor is provided, which is encoded by the nucleic acid sequence of SEQ JD NO. 1 or variants thereof, wherein the variants are each defined as having one or more substitutions, insertions and/or deletions as compared to the sequence of SEQ ID NO. 1, provided that said variants hybridize under moderately stringent conditions to a nucleic acid which comprises the sequence of SEQ ID NO. 1 and further provided that said variants code for a protein λvith activity as pain receptor or provided that said variants comprise nucleic acid changes due to the degeneracy of the genetic code, which code for the same or a functionally equivalent amino acid as the nucleic acid sequence of SEQ ID NO.1.

According to a preferred embodiment, the invention also comprises a pain receptor or a fragment thereof, which is encoded by at least the sequence of nucleic acids No. 2001 - 2175 of SEQ ID NO. 1 or valiants thereof, wherein the variants are each defined as having one or more substitutions, insertions and/or deletions as compared to the sequence of nucleic acids No. 2001 - 2175 of SEQ ID NO. 1, provided that said variants hybridize under moderately stringent conditions to a nucleic acid which comprises the sequence of nucleic acids No. 2001 - 2175 of SEQ ID NO. 1 or provided that said variants comprise nucleic acid changes due to the degeneracy of the genetic code, which code for the same or a functionally equivalent amino acid as the nucleic acid sequence of nucleic acids No. 2001 - 2175 of SEQ ID NO. 1.

Furthermore, the present invention provides an isolated nucleic acid which comprises the sequence of SEQ ID NO. 1 or variants thereof, wherein the variants are each defined as having one or more substitutions, insertions and/or deletions as compared to the sequence of SEQ ID NO. 1, provided that said variants hybridize under moderately stringent conditions to a nucleic acid which comprises the sequence of SEQ ID NO. 1, and further provided that said variants code for a protein with activity as pain receptor or provided that said variants comprise nucleic acid changes due to the degeneracy of the genetic code, which code for the same or a functionally equivalent amino acid as the nucleic acid sequence of SEQ ID NO.l. This isolated nucleic acid preferably comprises at least the sequence of nucleic acids No. 2001 - 2175 of SEQ ID NO. 1 or variants thereof, wherein the variants are each defined as having one or more substitutions, insertions and/or deletions as compared to the sequence of nucleic acids No. 2001 - 2175 of SEQ ID NO. 1, provided that said variants hybridize under moderately stringent conditions to a nucleic acid which comprises the sequence of nucleic acids No. 2001 - 2175 of SEQ ID NO. 1 or provided that said variants comprise nucleic acid changes due to the degeneracy of the genetic code, which code for the same or a functionally equivalent amino acid as the nucleic acid sequence of nucleic acids No. 2001 - 2175 of SEQ LD NO. 1. The above mentioned isolated nucleic acids may further comprise a nucleic acid encoding a tag polypeptide covalently linked thereto or a nucleic acid specifying one or more regulatory sequences operably linked thereto.

According to a further embodiment, the nucleic acids of the present invention comprise transcriptional products of one of the above nucleic acids, e.g. mRNA, as well as nucleic acids, which selectively hybridize to said transcriptional products of the nucleic acids under moderate stringent conditions.

The term "nucleic acid sequence" refers to a heteropolymer of nucleotides or the sequence of these nucleotides. The terms "nucleic acid" and "polynucleotide" are used interchangeably herein to refer to a heteropolymer of nucleotides.

The polynucleotides of the present invention also include, but are not limited to, a polynucleotide that hybridizes to the complement of the disclosed nucleotide sequences under moderately stringent or stringent hybridization conditions; a polynucleotide which is an allelic variant of any polynucleotide recited above; a polynucleotide which encodes a species homologue of any of the herein disclosed proteins; or a polynucleotide that encodes a polypeptide comprising an additional specific domain or truncation of the disclosed proteins.

Stringency of hybridization, as used herein, refers to conditions under which polynucleotide duplexes are stable. As known to those of skill in the art, the stability of duplex is a function of sodium ion concentration and temperature (see, for example, Sambrook et al., Molecular Cloning: A Laboratoiy Manual 2^nd Ed. (Cold Spring Harbor Laboratory, (1989)). Stringency levels used to hybridize can be readily varied by those of skill in the art.

The phrase "low stringency hybridization" refers to conditions equivalent to hybridization in 10% formamide, 5 x Denhart's solution, 6 x SSPE, 0.2% SDS at 42°C, followed by washing in 1 x SSPE, 0.2% SDS, at 50°C. Denhart's solution and SSPE (see, e.g., Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1989) are well known to those of skill in the art as are other suitable hybridization buffers.

As used herein, the phrase "moderately stringent hybridization" refers to conditions that permit DNA to bind a complementary nucleic acid that has about 60% identity, preferably about 75% identity, more preferably about 85% identity to the DNA; with greater than about 90% identity to said DNA being especially preferred. Preferably, moderately stringent conditions are conditions equivalent to hybridization in 50% formamide, 5 x Denhart's solution, 5 x SSPE, 0.2% SDS at 42°C, followed by washing in 0.2 x SSPE, 0.2% SDS, at 65°C.

The phrase "high stringency hybridization" refers to conditions that permit hybridization of only those nucleic acid sequences that form stable duplex in 0.018M NaCl at 65°C. (i.e., if a duplex is not stable in 0.018M NaCl at 65.degree°C, it will not be stable under high stringency conditions, as contemplated herein). High stringency conditions can be provided, for example, by hybridization in 50% formamide, 5 x Denhart's solution, 5 x SSPE, 0.2% SDS at 42°C, followed by washing in 0.1 x SSPE, and 0.1% SDS at 65°C.

Further, nucleic acid hybridization techniques can be used to identify and obtain a nucleic acid within the scope of the invention. Briefly, any nucleic acid having some homology to a sequence set forth in this invention, or fragment thereof, can be used as a probe to identify a similar nucleic acid by hybridization under conditions of moderate to high stringency. Such similar nucleic acid then can be isolated, sequenced, and analyzed to determine whether they are within the scope of the invention as described herein.

Hybridization can be done by Southern or Northern analysis to identify a DNA or RNA sequence, respectively, that hybridizes to a probe. The probe can be labeled with a radioisotope such as ³²P, an enzyme, digoxygenin, or by biotinylation.

The DNA or RNA to be analyzed can be electrophoretically separated on an agarose or polyacrylamide gel, transferred to nitrocellulose, nylon, or other suitable membrane, and hybridized with the probe using standard techniques well known in the art such as those described in sections 7.39-7.52 of Sambrook et al., (1989) Molecular Cloning, 2^nd edition, Cold Spring Harbor Laboratory, Plainview, NY.

Typically, a probe is at least about 20 nucleotides in length. For example, a probe corresponding to a 20 nucleotide sequence set forth in this invention can be used to identify a nucleic acid identical to or similar to a nucleic acid sequence set forth in the group of nucleic acids of the present invention. In addition, probes longer or shorter than 20 nucleotides can be used.

Any cell containing an isolated nucleic acid within the scope of the invention is itself within the scope of the invention. This includes, without limitation, prokaryotic and eukaryotic cells. It is noted that cells containing an isolated nucleic acid of the invention are not required to express the isolated nucleic acid. In addition, the isolated nucleic acid can be integrated into the genome of the cell or maintained in an episomal state. In other words, cells can be stably or transiently transfected with an isolated nucleic acid of the invention.

Generally, the term "nucleic acid" as used herein encompasses both RNA and DNA, including cDNA, genomic DNA, and synthetic (e. g., chemically synthesized) DNA.

The nucleic acid can be double-stranded or single-stranded. Where single-stranded, the nucleic acid can be the sense strand or the antisense strand. In addition, nucleic acid can be circular or linear.

The term "isolated" as used herein with reference to nucleic acid refers to a naturally- occurring nucleic acid that is not immediately contiguous with both of the sequences with which it is immediately contiguous (one on the 5'end and one on the 3 'end) in the naturally-occurring genome of the organism from which it is derived.

For example, an isolated nucleic acid can be, without limitation, a recombinant DNA molecule of any length, provided one of the nucleic acid sequences normally found immediately flanking that recombinant DNA molecule in a naturally-occurring genome is removed or absent. Thus, an isolated nucleic acid includes, without limitation, a recombinant DNA that exists as a separate molecule (e. g., a cDNA or a genomic DNA fragment produced by PCR or restriction endonuclease treatment) independent of other sequences as well as recombinant DNA that is incorporated into a vector, an autonomously replicating plasmid, a virus (e. g., a retrovirus, adenovirus, or herpes virus), or into the genomic DNA of a prokaryote or eukaryote. In addition, an isolated nucleic acid can include a recombinant DNA molecule that is part of a hybrid or fusion nucleic acid sequence.

The term "isolated" also includes any non-naturally-occurring nucleic acid since non- naturally-occurring nucleic acid sequences are not found in nature and do not have immediately contiguous sequences in a naturally-occurring genome. For example, non- naturally-occurring nucleic acid such as an engineered nucleic acid is considered to be isolated nucleic acid. Engineered nucleic acid can be made using common molecular cloning or chemical nucleic acid synthesis techniques. Isolated non-naturally-occurring nucleic acids can be independent of other sequences, or incorporated into a vector, an autonomously replicating plasmid, a virus (e. g., a retrovirus, adenovirus, or herpes virus), or the genomic DNA of a prokaryote or eukaryote. In addition, a non-naturally-occurring nucleic acid can include a nucleic acid molecule that is part of a hybrid or fusion nucleic acid sequence.

It will be apparent to those of skill in the art that a nucleic acid existing among hundreds to millions of other nucleic acid molecules within, for example, cDNA or genomic libraries, or gel slices containing a genomic DNA restriction digest is not to be considered an isolated nucleic acid.

According to a further embodiment, the nucleic acids of the present invention provide anti- sense DNA or RNA. Antisense DNA or RNA molecules bind specifically with a targeted RNA message, interrupting the expression of the mRNA product. The antisense binds to the messenger RNA forming a double stranded molecule that cannot be translated by the cell. Typically, an antisense oligonucleotide is about 15-25 nucleotides in length. In addition, chemically reactive groups, such as iron-linked ethylenediaminetetraacetic acid (EDTA-Fe), can be attached to an antisense oligonucleotide, causing cleavage of the mRNA at the site of hybridization. These and other uses of antisense methods to inhibit the translation of nucleic acid are well known in the art (Marcus -Sakura, Anal. Biochem, 172: 289 (1988)).

The present invention further provides pain receptor proteins, which comprise the arnino acid sequence of SEQ JD NO. 2 or a variant of said sequence, wherein said variant comprises one or more insertions, substitutions and/or deletions as compared to the sequence of SEQ ID NO. 2, and wherein the biological activity as pain receptor is substantially equal to the activity of the pain receptor comprising the unmodified arnino acid sequence of SEQ ID NO. 2.

According to a preferred embodiment, the invention provides a pain receptor protein or a fragment thereof, which comprises at least the amino acid sequence of amino acids NO. 667 - 724 of SEQ ID NO. 2 or a variant of said sequence, wherein said variant comprises one or more insertions, substitutions and/or deletions as compared to the sequence of amino acids NO. 667 - 724 of SEQ ID NO. 2.

The amino acid sequences of the fusion proteins of the present invention also encompass all sequences differing from the herein disclosed sequences by amino acid insertions, deletions, and substitutions.

Preferably, amino acid "substitutions" are the result of replacing one amino acid with another amino acid having similar structural and/or chemical properties, i.e., conservative amino acid replacements. Amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues involved. For example, nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valrne, proline, phenylalanine, tryptophan, and methionine; polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine; positively charged (basic) amino acids include arginine, lysine, and histidine; and negatively charged (acidic) amino acids include aspartic acid and glutamic acid. Amino acid changes, which affect the N-terminal and C-terminal part of the protein or polypeptide, often do not change the protein activity, because these parts are often not involved in the biological activity. "Insertions" or "deletions" are typically in the range of about 1 to 5 amino acids. The variation allowed may be experimentally determined by systematically making insertions, deletions, or substitutions of amino acids in a polypeptide molecule using recombinant DNA techniques and assaying the resulting recombinant variants for activity. This does not require more than routine experiments for the skilled artisan.

It can be desired to eliminate one or more of the cysterns of the sequence, since cysteines can cause the unwanted formation of multimers when the protein is produced recombinant. Multimers may complicate purification procedures. Each of the suggested modifications is in range of the current state of the art, and under the retention of the biological activity of the protein products.

According to a further aspect of the invention, a vector is provided which comprises the above mentioned nucleic acids. Preferably, an expression vector is provided, which comprises said nucleic acids coding for a VRL3 receptor protein. This expression vector preferably comprises one or more regulatory sequences. The term "expression vector" generally refers to a plasmid or phage or virus or vector, for expressing a polypeptide from a DNA (RNA) sequence. An expression vector can comprise a transcriptional unit comprising an assembly of (1) a genetic element or elements having a regulatory role in gene expression, for example, promoters or enhancers, (2) a structural or coding sequence which is transcribed into mRNA and translated into protein, and (3) appropriate transcription initiation and termination sequences. Structural units intended for use in yeast or eukaryotic expression systems preferably include a leader sequence enabling extracellular secretion of translated protein by a host cell. Alternatively, where recombinant protein is expressed without a leader or transport sequence, it may include an N-terminal methionine residue. This residue may or may not be subsequently cleaved from the expressed recombinant protein to provide a final product.

The present invention further provides hosts, e.g. host cells, which have been transformed to contain the polynucleotides of the invention. The term "transformation" means introducing DNA into a suitable host cell so that the DNA is replicable, either as an extrachromosomal element, or by chromosomal integration. For example, such host cells may contain nucleic acids of the invention introduced into the host cell using known transformation methods. The present invention still further provides host cells genetically engineered to express the polynucleotides of the invention, wherein such polynucleotides are in operative association with a regulatory sequence heterologous to the host cell which drives expression of the polynucleotides in the cell.

The host cell can be a higher eukaryotic host cell, such as a mammalian cell, a lower eukaryotic host cell, such as a yeast cell, or can be an insect cell, or the host cell can be a prokaryotic cell, such as a bacterial cell. Introduction of the recombinant construct into the host cell can be effected by calcium phosphate transfection, DEAE, dextran mediated transfection, or electroporation (Davis, L. et al., Basic Methods in Molecular Biology (1986)).

The most preferred cells are those which do not normally express the particular polypeptide or protein or which expresses the polypeptide or protein at low natural level. Mature proteins can be expressed in mammalian cells, yeast, bacteria, or other cells under the control of appropriate promoters. Appropriate cloning and expression vectors for use with prokaryotic and eukaryotic hosts are described by Sambrook, et al., in Molecular Cloning: A Laboratory Manual, 2^nd Edition, Cold Spring Harbor, New York (1989).

The mammalian cell is preferably a CHO, COS, HeLa, 293T, HEH or BHK cell. As procaryotic cells, E.coli and Bacillus subtilis are preferred. Also, derivatives of E. coli can be used (TOP 10, TOPI OF' and GI826 of Invitrogen; DH5alpha, HB101, JM109, BMH 71- 18 mutS and BL21(DE3)pLysS of Promega, DH5alphaF' of Teknova, Half Moon Bay, CA; JM101 (ATCC 33876)).

Suitable Bacillus subtilis strains are, e.g., Bacillus subtilis (Ehrenberg) Cohn strains (ATCC 82, 82D, 465, 4529, 4944, 6051, 6051-U, 6051 -A, 6455).

Suitable yeast cells are, e.g., Saccharomyces cerevisiae Hansen cells (ATCC 287) and derivatives thereof. Alternatively, the protein according to the invention can be produced in transgenic plants (e.g. potatoes, tobacco).

According to a further embodiment, a transgenic non-human animal is provided comprising an isolated nucleic acid encoding the pain receptor of the invention. Transgenic animals can be aquatic animals (such as fish, sharks, dolphin, and the like), farm animals (such as pigs, goats, sheep, cows, horses, rabbits, and the like), rodents (such as rats, guinea pigs, and mice), non-human primates (such as baboons and chimpanzees), and domestic animals (such as dogs and cats).

Several techniques known in the art can be used to introduce nucleic acid into animals to produce the founder lines of transgenic animals. Such techniques include, without limitation, pronuclear microinjection, retrovirus mediated gene transfer into germ lines (Nan der Putten et al, Proc. Νatl. Acad. Sci, USA, 82: 6148-6152 (1985)), gene transfection into non human embryonic stem cells (Gossler A et al, Proc Νatl Acad Sci USA 83: 9065-9069 (1986)), gene targeting into non human embryonic stem cells (Thompson et al., Cell, 56: 313-321 (1989)), nuclear transfer of somatic nuclei (Schnieke AE et al., Science 278: 2130-2133 (1997)), and electroporation of embryos.

For a review of techniques that can be used to generate and assess transgenic animals, skilled artisans can consult Gordon (Intl. Rev. Cytol., 115: 171-229 (1989)), and may obtain additional guidance from, for example: Hogan et al., "Manipulating the Mouse Embryo"Cold Spring Harbor Press, Cold Spring Harbor, Y (1986); Krimpenfort et al., BiolTechnology, 9: 844-847 (1991); Palmiter et al., Cell, 41 : 343-345 (1985); Kraemer et al. /'Genetic Manipulation of the Early Mammalian Embryo "Cold Spring Harbor Press, Cold Spring Harbor, ΝY (1985); Hammer et al., Nature, 315: 680-683 (1985).

According to a further preferred embodiment the present invention provides an antibody or an aptamer, which is directed against a pain receptor protein or a mutant, variant or fragment of any of said receptor protein, as disclosed above.

The term "antibody" as used herein refers to intact antibodies as well as antibody fragments that retain some ability to selectively bind an epitope. Such fragments include, without limitation, Fab, F (ab') 2, and Fv antibody fragments. The term "epitope" refers to an antigenic determinant on an antigen to which the paratope of an antibody binds. Epitopic determinants usually consist of chemically active surface groupings of molecules (e. g., amino acid or sugar residues) and usually have specific three dimensional structural characteristics as well as specific charge characteristics.

Any antibody having specific binding affinity for an amino acid encoded by a nucleic acid within the scope of the invention is itself within the scope of the invention. Thus, any monoclonal or polyclonal antibody having specific binding affinity for an herein defined amino acid sequence is within the scope of the invention.

Antibodies within the scope of the invention can be prepared using any method. For example, any substantially pure prptein provided herein, or fragment thereof, can be used as an immunogen to elicit an immune response in an animal such that specific antibodies are produced. Thus, an intact full-length protein or fragments containing small peptides can be used as an immunizing antigen. In addition, the immunogen used to immunize an animal can be chemically synthesized or derived from translated cDNA. Further, the immunogen can be conjugated to a carrier polypeptide, if desired. Commonly used carriers that are chemically coupled to an immunizing polypeptide or protein include, without limitation, keyhole limpet hemocyanin (KLH), thyroglobulin, bovine serum albumin (BSA), and tetanus toxoid.

The preparation of polyclonal antibodies is well-known to those skilled in the art. See, e. g., Green et al., Production of Polyclonal Antisera, in Immunochemical Protocolls (Manson, ed.), pages 1-5 (Humana Press 1992) and Coligan et al., Production of Polyclonal Antisera in Rabbits, Rats, Mice and Hamsters, in Current Protocolls In Immunology, section 2.4.1 (1992). In addition, those of skill in the ait will know of various techniques common in the immunology arts for purification and concentration of polyclonal antibodies, as well as monoclonal antibodies (Coligan, et al., Unit 9, Current Protocols in Immunology, Wiley Interscience, 1994).

The preparation of monoclonal antibodies also is well-known to those skilled in the ait. See, e. g., Kohler & Milstein, Nature 256: 495 (1975); Coligan et al., sections 2.5.1-2.6.7; and Harlow et al., Antibodies: A Laboratory Manual, page 726 (Cold Spring Harbor Pub. 1988). Briefly, monoclonal antibodies can be obtained by injecting mice with a composition comprising an antigen, verifying the presence of antibody production by analyzing a serum sample, removing the spleen to obtain B lymphocytes, fusing the B lymphocytes with myeloma cells to produce hybridomas, cloning the hybridomas, selecting positive clones that produce antibodies to the antigen, and isolating the antibodies from the hybridoma cultures. Monoclonal antibodies can be isolated and purified from hybridoma cultures by a variety of well-established techniques. Such isolation techniques include affinity chromatography with Protein-A Sepharose, size-exclusion chromatography, and ion-exchange chromatography. See, e. g., Coligan et al., sections 2.7.1-2.7.12 and sections Immunoglobulin G (IgG), in Methods In Molecular Biology, Vol. 10, pages 79-104 (Humana Press 1992).

In addition, methods of in vitro and in vivo multiplication of monoclonal antibodies are well-known to those skilled in the art. Multiplication in vitro can be carried out in suitable culture media such as Dulbecco's Modified Eagle Medium or RPMI 1640 medium, optionally replenished by mammalian serum such as fetal calf serum, or trace elements and growth-sustaining supplements such as normal mouse peritoneal exudate cells, spleen cells, and bone marrow macrophages. Production in vitro provides relatively pure antibody preparations and allows scale-up to yield large amounts of the desired antibodies. Large scale hybridoma cultivation can be carried out by homogenous suspension culture in an airlift reactor, in a continuous stirrer reactor, or in immobilized or entrapped cell culture. Multiplication in vivo may be carried out by injecting cell clones into mammals histocompatible with the parent cells (e. g., osyngeneic mice) to cause growth of antibody- producing tumors.

Optionally, the animals are primed with a hydrocarbon, especially oils such as pristane (tetramethylpentadecane) prior to injection. After one to three weeks, the desired monoclonal antibody is recovered from the body fluid of the animal.

The antibodies within the scope of the invention also can be made using non-human primates. General techniques for raising therapeutically useful antibodies in baboons can be found, for example, in Goldenberg et al., International Patent Publication WO 91/11465 (1991) and Losman et al., Int. J Cancer 46: 310 (1990).

Alternatively, the antibodies can be "humanized" monoclonal antibodies. Humanized monoclonal antibodies are produced by transferring mouse complementarity determining regions (CDRs) from heavy and light variable chains of the mouse immunoglobulin into a human variable domain, and then substituting human residues in the fi-amework regions of the murine counterparts. The use of antibody components derived from humanized monoclonal antibodies obviates potential problems associated with the immunogenicity of murine constant regions when treating humans. General techniques for cloning murine immunoglobulin variable domains are described, for example, by Orlandi et al., Proc. Nat'l. Acad. Sci.USA 86: 3833 (1989). Techniques for producing humanized monoclonal antibodies are described, for example, by Jones et al., Nature 321: 522 (1986); Riechmann et al., Nature 332: 323 (1988); Verhoeyen et al., Science 239: 1534 (1988); Carter et al., Proc. Nat'l. Acad. Sci. USA 89: 4285 (1992); Sandhu, Crit. Rev. Biotech. 12: 437 (1992); and Singer et al., J : Immunol. 150: 2844 (1993).

Antibodies of the present invention also may be derived from human antibody fragments isolated from a combinatorial immunoglobulin library. See, for example, Barbas et al., Methods: A Companion To Methods In Enzymology, Vol. 2, page 119 (1991) and Winter et al., Ann. Rev. Immunol. 12: 433 (1994). Cloning and expression vectors that are useful for producing a human immunoglobulin phage library can be obtained, for example, from STRATAGENE Cloning Systems (La Jolla, CA, USA).

In addition, antibodies of the present invention may be derived from a human monoclonal antibody. Such antibodies are obtained from transgenic mice that have been "engineered" to produce specific human antibodies in response to antigenic challenge. In this technique, elements of the human heavy and light chain loci are introduced into strains of mice derived from embryonic stem cell lines that contain targeted disruptions of the endogenous heavy and light chain loci. The transgenic mice can synthesize human antibodies specific for human antigens and can be used to produce human antibody-secreting hybridomas. Methods for obtaining human antibodies from transgenic mice are described by Green et al., Nature Genet. 7: 13 (1994); Lonberg et al., Nature 368: 856 (1994); and Taylor et al., Int. Immunol. 6: 579 (1994).

All mentioned antibodies may additionally be linked to a toxic agent and/or to a detectable agent.

Alternatively, the present invention provides aptamers, which are directed against a pain receptor protein of the present invention or a mutant, fragment or variant thereof. Aptamers are DNA or RNA molecules that have been selected from random pools based on their ability to bind other molecules. Aptamers have been selected which bind nucleic acid, proteins, small organic compounds, and even entire organisms.

Additionally a hybridoma is provided by the present invention, which produces a monoclonal antibody having binding specificity to any one of the pain receptors or variants or fragments thereof. For methods of producing those hybridomas, see the above definitions on how to produce antibodies.

According to a preferred embodiment, an inhibitor of the pain receptor of the pain receptor is provided, wherein said inhibitor is selected from the group consisting of a molecule that reduces the level of mRNA encoding said pain receptor polypeptide, a molecule that reduces the level of said pain receptor polypeptide, and a molecule that reduces the biological activity of said pain receptor. The inhibitor may be selected from the group consisting of an antisense nucleic acid, a ribozyme, double stranded RNA, an antibody, a peptide and a peptidomimetic or small molecules which bind to the receptor protein of the present invention but do not elicit a response, so that the activity of the protein is prevented.

According to a further preferred embodiment, the invention provides an agonist of the herein disclosed pain receptor. An agonist is a signalling molecule (hormone, neurotransmitter or synthetic drug) which binds to a receptor, inducing a conformational change which produces a response such as contraction, relaxation, secretion, change in enzyme activity, etc. The term „receptor agonist" as used herein generally means any substance, which shows an affinity to the pain receptor and has an intrinsic activity. The term intrinsic activity" is widely used as an empirical measure of the maximal response to a test agonist as a fraction of that to a full agonist acting at the same receptor type. A drug with an intrinsic activity of less than 1 is behaving as a partial agonist in the preparation concerned. Full agonism is not an absolute state but is both ligand- and preparation-dependent (i.e. a full agonist in one tissue might behave as a partial agonist in a preparation with a lower receptor reserve). Full agonists as well as partial agonists are envisioned by the present invention.

The present invention fuither provides compositions, comprising one or more kinds of nucleic acids of the invention, one or more of the receptor proteins disclosed herein, the inhibitors/agonists as mentioned above or one or more kinds of antibodies of the invention, optionally in combination with a pharmaceutically acceptable carrier and/or diluent.

The ingredients of the present invention are preferably used in form of a pharmaceutical composition where they are mixed with suitable carriers or excipients in doses to treat or ameliorate the disease. Such a composition may also contain (in addition to the ingredient and the carrier) diluents, fillers, salts, buffers, stabilizers, solubilizers and other materials well known in the art. The term "pharmaceutically acceptable" means a non-toxic material that does not interfere with the effectiveness of the biological activity of the active ingredient(s). The characteristics of the carrier will depend on the route of administration. The pharmaceutical composition may further contain other agents which either enhance the activity of the activity or use in treatment. Such additional factors and/or agents may be included in the pharmaceutical composition to produce a synergistic effect or to minimize side-effects.

Techniques for formulation and administration of the compounds of the instant application may be found in "Remington's Pharmaceutical Sciences", Mack Publishing Co., Easton, PA, latest edition. Whenever the compositions are to be used for medical purposes, they will contain a therapeutically effective dose of the respective ingredient. A therapeutically effective dose further refers to that amount of the compound/ingredient sufficient to result in amelioration of symptoms, e.g., treatment, healing, prevention or amelioration of such conditions. In the context of the present invention, a therapeutically effective dose is to be understood as an amount of the compound/ingredient, which results in a statistically significant reduction of pain sensation. Suitable routes of adminisfration may, for example, include oral, rectal, transmucosal, or intestinal administration; parenteral delivery, including intramuscular, subcutaneous, intrameduUary injections, as well as intrathecal, direct rntraventricular, intravenous, intraperitoneal or intranasal injections. Administration of the antibody of the present invention used in the pharmaceutical composition of the present invention can be carried out in a variety of conventional ways, such as oral ingestion, inhalation, topical application or cutaneous, subcutaneous, intraperitoneal, parenteral or intravenous injection. Intravenous administration to the patient is preferred.

A typical composition for intravenous infusion can be made up to contain 250 ml of sterile Ringer's solution, and 10 mg of antibody. See Remington's Pharmaceutical Science (15^th Ed., Mack Publishing Company, Easton, Ps., 1980).

The above mentioned compositions may also be used as diagnostic compositions.

The invention also relates to methods for producing a substantially pure protein comprising growing a culture of the cells of the invention in a suitable culture medium, and purifying the protein from the culture. For example, the methods of the invention include a process for producing a protein in which a host cell containing a suitable expression vector that includes a protein of the invention is cultured under conditions that allow expression of the encoded protein. The protein can be recovered from the culture, conveniently from the culture medium, and can be further purified. The resulting expressed protein may for example be purified from such culture (i.e., from culture medium or cell extracts) using known purification processes, such as gel filtration and ion exchange chromatography.

Furthermore, according to an embodiment of the invention, a method of detection of the expression level of the transcriptional products of the nucleic acids of the present invention in a mammal is provided, said method comprising (a) obtaining a biological sample from said mammal and (b) contacting said biological sample with a reagent which detects said transcriptional products. Preferably, this reagent is a nucleic acid and is even more preferably detectably labelled (for further information, see also chapter "probes", above). If so, the expression level of the transcriptional products may be detected by detecting in a sample a mRNA transcript that encodes the pain receptor as disclosed herein, said method comprising the steps of (a) contacting said sample under moderately stringent hybridizing conditions with the nucleic acid disclosed herein to form a duplex; and (b) detecting the presence of said duplex.

Analogously, a method of detection of the expression level of the pain receptor protein of the invention is provided, said method comprising (a) obtaining a biological sample from said mammal and (b) contacting said biological sample with a reagent which detects the pain receptor protein of the invention. According to a preferred embodiment, said reagent is an antibody as herein defined, which is more preferably detectably labelled.

According to a further embodiment, a method of detection of a mutation in a nucleic acid of the invention is provided, said method comprising (a) obtaining a biological sample from said mammal and (b) contacting said biological sample with a reagent which detects the presence or absence of a mutation in one of said nucleic acid. Preferably, said reagent is a nucleic acid, which more preferably is detectably labelled.

Detectable labellings in accordance with the present invention are, for example, radioactive labelling, fluorescent labelling, biotin, digoxigenin or peroxidase labellings or a labelling, which is detectable by means of alkaline phosphatase.

Furthermore, the present invention provides a method of detection of a mutation in a pain receptor protein according to the invention, said method comprising (a) obtaining a biological sample from said mammal and (b) contacting said biological sample with a reagent which detects the presence or absence of a mutation in said protein. According to a preferred embodiment, said reagent is an antibody, which is more preferably detectably labelled.

According to a preferred embodiment, the present invention provides a method of pain inhibition comprising administering an effective pain inhibiting amount of the above defined therapeutic compositions to a patient in need of such treatment. Preferably, these compositions comprise antibodies, antisense DNA/RNA or most preferably an inhibitor of the pain receptor as defined above. For ways of administration see above.

Alternatively, an effective pain inhibiting amount of the composition containing an pain receptor agonist may be administered to a patient in need of such treatment over a prolonged time period. This therapeutic approach is in particular suitable for the treatment of chronic pain conditions and leads to a desensitization of the pain receptors as defined herein.

The physician in any event will determine the actual dosage which will be most suitable for an individual patient and will vary with the age, weight and response of the particular patient. There can, of course, be individual instances where higher or lower dosage ranges are merited, and such are within the scope of this invention.

It is desirous to devise screening methods to identify compounds which stimulate or which inhibit the function of the pain receptor. Accordingly, in a further aspect, the present invention provides for a method of screening compounds to identify those which stimulate or which inhibit the function of the receptor. In general, agonists or inhibitors may be employed for therapeutic and prophylactic purposes. Compounds may be identified from a variety of sources, for example, cells, cell-free preparations, chemical libraries, and natural product mixtures. Such agonists or inhibitors so-identified may be natural or modified substrates, ligands etc., as the case may be, of the receptor; or may be structural or functional mimetics thereof (see Coligan et al., Current Protocols in Immunology 1(2): Chapter 5 (1991)).

Therefore, the present invention provides a method of identifying/screening compounds that bind to the pain receptor of the present invention. According to one preferred embodiment, this method comprises the steps of generating the pain receptor protein of the invention, contacting said protein with a candidate compound, measuring the binding of said compound by a suitable detection method, selecting a compound that has been tested positive, optionally repeating steps the latter steps with a suitably modified form of the positively tested compound. The screening method may simply measure the binding of a candidate compound to the receptor protein, or to cells or membranes bearing the receptor protein, by means of a label directly or indirectly associated with the candidate compound. Alternatively, the screening method may involve competition with a labeled competitor. Further, these screening methods may test whether the candidate compound results in a signal generated by activation or inhibition of the protein, using detection systems appropriate to the cells bearing the receptor protein. Inhibitors of activation are generally assayed in the presence of a known agonist and the effect on activation by the agonist by the presence of the candidate compound is observed. Constitutively active proteins may be employed in screening methods for inverse agonists or inhibitors, in the absence of an agonist or inhibitor, by testing whether the candidate compound results in inhibition or activation of the protein.

According to a preferred embodiment, a method of screening for agonists and/or antagonists of the pain receptor of the invention is provided, the method comprising the steps of: a) transfecting any one of the above defined host cells with a nucleic acid or a vector in accordance with the invention; b) contacting a candidate compound with said transfected host cells; c) determining, whether the candidate compound modifies the inflow or outflow of Ca ⁺ in said host cell by an agent suitable for detecting the Ca²⁺ level in said host cell; d) selecting a compound that has been tested positive in step (c), e) optionally repeating steps (a) - (d) with a suitably modified form of the compound of step (d).

Preferably, the agent for detecting the Ca²⁺ level in said host cell is a fluorescent indicator, e.g. fura-2 or aequorin.

Furthermore, the present invention provides a kit comprising at least one of

(a) a nucleic acid molecule,

(b) a vector,

(c) a host, (d) a protein,

(e) an antibody or aptamer,

(f) an inhibitor,

(g) an agonist, as they have been defined herein above.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

The invention is now further illustrated by the accompanying drawings, in which:

Fig. 1 shows the complete coding sequence and predicted protein sequence of the pain receptor of the present invention.

Fig. 2 shows a multiple sequence alignment (ClustalW, default parameters) of the four human members of the NR protein family. Alignment was drawn with "Boxshade 3.21" (http://www.ch.embnet.org/software/BOX_fomi.html). Identical and conserved residues are marked by black and grey background, respectively.

Fig. 3 shows a dendrogram (ClustalX, default parameters) comparing the known human VR family members. All four members are nearly equally related to each other, there are no clear subbranches in this family. The novel member cloned here is indicated by an arrow, hs = homo sapiens.

Fig. 4 shows the issue expression of human NRL3. RT-PCR analysis of selected adult tissues (Clontech). A weak signal could be detected after 35 cycles in 3 = placenta, 6 = skeletal muscle, 8 = pancreas, 9 = spleen, 10 = thymus, 16 == leukocytes. No expression could be detected in 1 = heart, 2 = brain, 4 = lung, 5 = liver, 7 = kidney, 11 = prostata, 12 = testis, 13 = ovary, 14 = small intestine, 15 = colon. No expression was observed for a control cDNA (17) nor for genomic DNA (18), a strong signal was detected for a VRL3 positive cDNA control (19). 20 is molecular weight marker. A positive control G3PDH with a house keeping gene showed signals in all tissues (data not shown).

Methods and Results

Starting with the published protein sequences of NRl and VRLl we performed BLAST searches with the TBLASTΝ algorithm against the public dbEST and HTGS databases at ΝCBI (http://www.ncbi.nlm.nih.gov/BLAST). No evidence for a novel NR homolog could be detected in dbEST, yet, a significant homology to NRl was identified on the genomic clone AC025125. Computer based gene prediction and sequence alignments allowed to predict a partial open reading frame of a novel NR homolog, which we designated NRL3.

To prove the existence of this gene, we designed primers from the predicted sequence and performed PCR amplification on retinal cDΝA (Clontech). PCR fragments were gel-eluted and directly sequenced by standard techniques. Sequencing results confirmed the presence of NRL3 and enabled us to fill gaps not predicted by computer analyses. To identify the complete coding region, we performed RACE PCR on retinal cDΝA (Clontech, Marathon Kit). By this we were able to identify the putative complete coding sequence with a start methionine preceded by inframe stop codons and an stop codon after an open reading frame of 2172 base pairs. The complete coding sequence and the predicted protein sequence is shown in figure 1.

NRL3 shows an overall homology to NRl, VRLl and NR-OAC of about 62%, 56% and 62%, respectively. Structure and domain predictions using various public databases demonstrated that NRL3 has the typical structure of NR proteins with six transmembrane domains, a pore loop between the fifth and sixth domain and three Ν-terminal ankyrin repeats. The multiple sequence alignment is shown in figure 2.

Further comparison with the other NR members showed that all four NRs are nearly equally related to each other and that there do not seem to be distinct subbranches within the NR family (figure 3).

To analyze the tissue distribution of NRL3 we performed northern blotting (Clontech multiple tissue northern blots), yet, we were not able to identify signals even after long exposure times suggesting a specific expression pattern (which is furthermore suggested by the fact that there is no EST of NRL3 in the public databases). We therefore performed RT-PCR analyses with selected adult tissues. We observed low expression of NRL3 (i. e. we needed a high number of PCR cycles) in a few analyzed tissues, i. e. placenta, skeletal muscle, pancreas, spleen, thymus, and leukocytes (figure 4).

Furthermore, the genomic structure of NRL3 was elucidated, which consists of 16 coding exons, and determined the chromosomal localization of NRL3 on the short aim of chromosome 17 (17pl3 near the marker D17S2042). Interestingly, it is most closely located to NRl making it tempting to speculate that both channels may be coordinately regulated.

References

1. Caterina, M.J. & Julius, D. Sense and specificity: a molecular identity for nociceptors. Curr Opin Neurobiol 9, 525-30 (1999).

2. McCleskey, E.W. & Gold, M.S. Ion channels of nociception. Annu Rev Physiol 61, 835- 56 (1999).

3. Caterina, M.J. et al. The capsaicin receptor: a heat-activated ion channel in the pain pathway. Nature 389, 816-24. (1997).

4. Caterina, M.J. et al. Impaired nociception and pain sensation in mice lacking the capsaicin receptor. Science 288, 306-13 (2000).

5. Davis, J.B. et al. Nanilloid receptor- 1 is essential for inflammatory thermal hyperalgesia. Nature 405, 183-7. (2000).

6. Caterina, M.J., Rosen, T.A., Tominaga, M., Brake, A.J. & Julius, D. A capsaicin- receptor homologue with a high threshold for noxious heat. Nature 398, 436-41. (1999).

7. Liedtke, W. et al. Nanilloid receptor-related osmotically activated channel (VR-OAC), a candidate vertebrate osmoreceptor. Cell 103, 525-35. (2000).

8. Strotmann, R., Harteneck, C, Νunnenmacher, K., Schultz, G. & Plant, T.D. OTRPC4, a nons elective cation channel that confers sensitivity to extracellular osmolarity. Nat Cell Biol 2, 695-702. (2000).

9. Szallasi, A. & Blumberg, P.M. Vanilloid (Capsaicin) receptors and mechanisms. Pharmacol Rev 51, 159-212. (1999).

10. Caterina, M. J. et al. Impaired nociception and pain sensation in mice lacking the capsaicin receptor. Science 288, 306 - 313 (2000).

Claims

PAIN RECEPTOR GENE AND PROTEINP A T E N T C L A I M S

1. A pain receptor, which is encoded by the nucleic acid sequence of SEQ ID No. 1 or variants thereof, wherein the valiants are each defined as having one or more substitutions, insertions and/or deletions as compared to the sequence of SEQ ID No. 1, provided that said variants hybridize under moderately stringent conditions to a nucleic acid which comprises the sequence of SEQ ID No. 1 and further provided that said variants code for a protein with activity as pain receptor or provided that said variants comprise nucleic acid changes due to the degeneracy of the genetic code, which code for the same or a functionally equivalent amino acid as the nucleic acid sequence of SEQ JD NO.1.

2. The pain receptor of claim 1 or a fragment thereof, which is encoded by at least the sequence of nucleic acids No. 2001 - 2175 of SEQ ID No. 1 or valiants thereof, wherein the variants are each defined as having one or more substitutions, insertions and/or deletions as compared to the sequence of nucleic acids No. 2001 - 2175 of SEQ JD No. 1, provided that said variants hybridize under moderately stringent conditions to a nucleic acid which comprises the sequence of nucleic acids No. 2001 - 2175 of SEQ ID No. 1 or provided that said variants comprise nucleic acid changes due to the degeneracy of the genetic code, which code for the same or a functionally equivalent amino acid as the nucleic acid sequence of nucleic acids No. 2001 - 2175 of SEQ ID No. 1.

3. An isolated nucleic acid which comprises the sequence of SEQ ID No. 1 or variants thereof, wherein the variants are each defined as having one or more substitutions, insertions and/or deletions as compared to the sequence of SEQ ID No. 1, provided that said variants hybridize under moderately stringent conditions to a nucleic acid which comprises the sequence of SEQ ID No. 1, and further provided that said variants code for a protein with activity as pain receptor or provided that said variants comprise nucleic acid changes due to the degeneracy of the genetic code, which code for the same or a functionally equivalent amino acid as the nucleic acid sequence of SEQ ID NO.1.

4. The isolated nucleic acid of claim 3, which comprises at least the sequence of nucleic acids No. 2001 - 2175 of SEQ JJD No. 1 or variants thereof, wherein the variants are each defined as having one or more substitutions, insertions and/or deletions as compared to the sequence of nucleic acids No. 2001 - 2175 of SEQ ID No. 1, provided that said variants hybridize under moderately stringent conditions to a nucleic acid which comprises the sequence of nucleic acids No. 2001 - 2175 of SEQ ID No. 1 or provided that said variants comprise nucleic acid changes due to the degeneracy of the genetic code, which code for the same or a functionally equivalent amino acid as the nucleic acid sequence of nucleic acids No. 2001 - 2175 of SEQ JD No. 1.

5. The isolated nucleic acid of claim 3 or 4, said isolated nucleic acid further comprising a nucleic acid encoding a tag polypeptide covalently linked thereto.

6. The isolated nucleic acid of claim 3 or 4, said isolated nucleic acid further comprising a nucleic acid specifying one or more regulatory sequences operably linked thereto.

7. A nucleic acid, which is a transcriptional product of one of the nucleic acids of claims 3 or 4.

8. A nucleic acid, which selectively hybridizes to transcriptional products of the nucleic acids of claims 3 or 4 under moderate stringent conditions.

9. The nucleic acid of claim 8, which is anti-sense DNA or RNA.

10. A pain receptor, which comprises the amino acid sequence of SEQ ID No. 2 or a variant of said sequence, wherein said variant comprises one or more insertions, substitutions and/or deletions as compared to the sequence of SEQ ID No. 2, and wherein the biological activity as pain receptor is substantially equal to the activity of the pain receptor comprising the unmodified amino acid sequence of SEQ ID No. 2.

11. The pain receptor of claim 10 or a fragment thereof, which comprises at least the amino acid sequence of amino acids No. 667 - 724 of SEQ ID No. 2 or a variant of said sequence, wherein said variant comprises one or more insertions, substitutions and or deletions as compared to the sequence of amino acids No. 667 - 724 of SEQ ID No. 2.

12. A vector, which comprises the nucleic acid sequence of any of claims 3-6.

13. An expression vector, which comprises the nucleic acid sequence of any of claims 3-6 and one or more regulatory sequences.

14. The vector of claim 12 or 13, which is a plasmid.

15. A host, which has been transformed with the vector of any of claims 12-14.

16. The host of claim 15, which is an eukaryotic cell.

17. The host of claim 16, which is a mammalian cell, plant cell, yeast cell or an insect cell.

18. The mammalian cell of claim 17, which is a CHO, COS, HeLa, 293T, HEH or BHK cell.

19. The host of claim 15, which is a prokaryotic cell.

20. The host of claim 19, which is E.coli or Bacillus subtilis.

21. A trans genie non-human animal comprising an isolated nucleic acid encoding the pain receptor of claim 1 , 2, 10 or 11.

22. An antibody or an aptamer, which is directed against a pain receptor or a mutant, variant or fragment of any of claims 1 , 2, 10 or 11.

23. The antibody of claim 22, wherein said antibody is selected from the group consisting of a polyclonal antibody, a monoclonal antibody, a humanized antibody, a chimeric antibody, and a synthetic antibody.

24. The antibody of claim 22 or 23 wherein said antibody is linked to a toxic agent and/or to a detectable agent.

25. A hybridoma which produces a monoclonal antibody having binding specificity to any one of the pain receptors or variants or fragments of claims 1, 2, 10 and 11.

26. An inhibitor of the pain receptor of claim 1, 2, 10 or 11, wherein said inhibitor is selected from the group consisting of a molecule that reduces the level of mRNA encoding said pain receptor protein, a molecule that reduces the level of said pain receptor protein, and a molecule that reduces the biological activity of said pain receptor.

27. The inhibitor of claim 26, wherein said inhibitor is selected from the group consisting of an antisense nucleic acid, a ribozyme, double stranded RNA, an antibody, a peptide and a peptidomimetic.

28. An agonist of the pain receptor of any of claims 1, 2, 10 or 11.

29. A composition, comprising the nucleic acid of any of claims 3, 4, 8 or 9 optionally in combination with a pharmaceutically acceptable carrier and or diluent.

30. A composition, comprising the protein of claim 1, 2, 10 or 11, optionally in combination with a pharmaceutically acceptable carrier and/or diluent.

31. A composition, comprising the antibody of any of claims 22-24, optionally in combination with a pharmaceutically acceptable carrier and/or diluent.

32. A composition, comprising the inhibitor of claim 26 or 27, optionally in combination with a pharmaceutically acceptable carrier and/or diluent.

33. A composition, comprising the agonist of claim 28, optionally in combination with a pharmaceutically acceptable carrier and/or diluent.

34. A composition of any of claims 29 - 33, which is a pharmaceutical composition.

35. A composition of any of claims 29 - 33, which is a diagnostic composition.

36. A method of making an isolated protein having the biological activity of the pain receptor of any of claims 1, 2, 10 or 11 comprising (a) culturing any one of the host cells of claim 15-20 under conditions such that the protein is expressed; and (b) recovering said protein.

37. A method of detection of the expression level of the transcriptional products of claim 7 in a mammal, said method comprising (a) obtaining a biological sample from said mammal and (b) contacting said biological sample with a reagent which detects the transcriptional products of claim 7.

38. The method of claim 37, wherein s aid reagent is a nucleic acid.

39. The method of claim 37 or 38, wherein said reagent is detectably labelled.

40. A method of detection of the expression level of the pain receptor protein of claim 1, 2, 10 or 11 in a mammal, said method comprising (a) obtaining a biological sample from said mammal and (b) contacting said biological sample with a reagent which detects the pain receptor protein of claim 1, 2, 10 or 11.

41. The method of claim 40, wherein said reagent is an antibody

42. The method of claim 40 or 41, wherein said reagent or said antibody is detectably labelled.

43. A method of detection of a mutation in a nucleic acid of claim 3 or 4 in a mammal, said method comprising (a) obtaining a biological sample from said mammal and (b) contacting said biological sample with a reagent which detects the presence or absence of a mutation in one of said nucleic acid.

44. The method of claim 43, wherein said reagent is a nucleic acid.

45. The method of claim 43, wherein said reagent is detectably labelled.

46. A method of detection of a mutation in a pain receptor protein of claim 1, 2, 10 or 11 in a mammal, said method comprising (a) obtaining a biological sample from said mammal and (b) contacting said biological sample with a reagent which detects the presence or absence of a mutation in said protein.

47. The method of claim 46, wherein said reagent is an antibody.

48. The method of claim 46, wherein said reagent is detectably labelled.

49. A method of pain inhibition comprising administering an effective pain inhibiting amount of the composition of any of claims 29 - 35 to a patient in need of such treatment.

50. The method of claim 49, wherein an effective pain inhibiting amount of the composition of claims 29-35 is administered to a patient in need of such treatment over a prolonged time period.

51. A method of identifying compounds that bind to pain receptor comprising the steps of

(a) generating the pain receptor protein of claim 1, 2, 10 or 11,

(b) contacting said protein with a candidate compound, (c) measuring the binding of said compound by a suitable detection method,

(d) selecting a compound that has been tested positive in step (c),

(e) optionally repeating steps (a) - (d) with a suitably modified form of the compound of step (d).

52. A kit comprising at least one of

(h) the nucleic acid molecule of any of claims 3 - 9

(i) the vector of any of claims 12 - 14

(j) the host of any of claims 15 - 20

(k) the protein of any of claims 1, 2, 10 or 11

(1) the antibody or aptamer of any of claims 22 - 24

(m)the inhibitor of claims 26 and 27

(n) the agonist of claim 28.

53. A method of screening for agonists and/or antagonists of the pain receptor of claim 1, 2, 10 or 11, the method comprising the steps of: f) transfecting any one of the host cells of claims 15 - 20 with a nucleic acid of claims 3 or 4 or a vector of claims 12-14; g) contacting a candidate compound with said transfected host cells; h) determining, whether the candidate compound modifies the inflow or outflow of Ca²⁺ in said host cell by an agent suitable for detecting the Ca ⁺ level in said host cell; i) selecting a compound that has been tested positive in step (c), j) optionally repeating steps (a) - (d) with a suitably modified form of the compound of step (d).

54. The method of claim 53, wherein the agent for detecting the Ca²⁺ level in said host cell is a fluorescent indicator.

55. The method of claim 54, wherein the fluorescent indicator is fura-2 or aequorin.