IE980956A1 - Nucleic Acid Encoding a Nervous Tissue Sodium Channel - Google Patents

Abstract

A novel nucleic acid sequence encoding for a mammalian voltage-gated, preferably TTX-resistant, sodium channel is isolated. Also disclosed are polypeptide products of recombinant expression of these sequences, expression vectors comprising the DNA sequence, and host cells transformed with these expression vectors. Other aspects of this invention are peptides whose sequences are based on the amino acid sequences deduced from these DNA sequences, antibodies specific for such proteins and peptides, procedures for detection and quantitation of such proteins and nucleic acids related thereto. Another aspect of this invention is the use of this voltage-gated, preferably tetrodotoxin-resistant, sodium channel as a therapeutic target for compounds.

Description

This invention relates generally to sodium channel proteins and more particularly to a novel nucleic acid sequence encoding for a mammalian α-subunit of a voltage-gated, preferably tetrodotoxin-resistant, nervous tissue sodium channel protein. This invention further relates to its production by recombinant technology.

The basic unit of information transmitted from one part of the nervous system to another is a single action potential or nerve impulse. The «transmission line“ for these impulses is the axon, or nerve fiber. The electrical excitability of the nerve membrane has been shown to depend on the membrane's voltage-sensitive ionic permeability system that allows it to use energy stored in ionic concentration gradients. Electrical activity of the nerve is triggered by a depolarization of the membrane, which opens channels through the membrane that are highly selective for sodium ions, which are then driven inward by the electrochemical gradient. Of the many ionic channels, the voltage-gated or voltage-sensitive sodium channel is one of the most studied. It is a transmembrane protein that is essentia] for the generation of action potentials in excitable cells. An excellent review of sodium channels is presented in Catterall, TINS 16(12), 500-506 (1993).

The cDNAs for several Na+ channels have been cloned and sequenced. Numa et al., Annals of the New York Academy of Sciences 479, 338-355 (1986), describe cDNA from the electric organ of eel and two different ones from rat brain. Rogart, U.S. Patent No. 5,380,836, describes cDNA from rat cardiac tissue. See also Rogart et al., Proc. Natl. Acad. Sci. 86, 8170-8174 (1989). The sequence of PN1 and its orthologs in humans (hNE) and rabbits (Na*s) have been published (see, for example, Klugbauer et al., EMBOJ 14,1084-1090 (1995) and Belcher et al., Proc. Natl. Acad. Sci. U.S.A. 923, 11034-11038 (1995)). The sequence of rat PN1 cloned from DRG and its function expression have been described (see, for example, Sangameswaran et al., J.Biol.Chem. 272,14805-14809 (1997)). Other cloned sodium channels include rat brain types I and Π, Noda et al., Nature 320,188-192 (1986), Ha, Auld et al., Neuron 1, 449-461 (1988), and HI, Kayano et al., FEBS Lett. 228, 187-194 (1988), rat γ I NT CL > i?/6. J ύ>ΤΗ 2ΐ|ΟΜ·?<ΡΤΚ iq-1^ -------- FT—! Ill II.....mi OPEN TO PUBLIC INSPECTION UMDFR SECTION 28 AML' · PULE 23 skeletal muscle (SkMl), Trimmer et al., Neuron 3, 33-49 (1989), rat NaCb6, Schaller et al., J. Neurosci. 15, 3231-3242 (1995), rat peripheral nerve sodium channel type 3 (rPN3), Sangameswaran et al., J. Biol Chem. 271, 5953-5956 (1996), also called SNS, Akopian et al.. Nature 379, 257-262 (1996), rat atypical channel, Felipe et al., J. Biol. Chem. 269, 301255 30131 (1994), and the rat glial sodium channel, Akopian et al., FEBS Lett. 400,183-187 (1997).

These studies have shown that the amino acid sequence of the Na+ channel has been conserved over a long evolutionary period. These studies have also revealed that the channel is a single polypeptide containing four internal repeats, or homologous domains (domains ΙΙΟ IV), having similar amino acid sequences. Each domain folds into six predicted and helical transmembrane segments: five are hydrophobic segments and one is highly charged with many positively charged lysine and arginine residues. This highly charged segment is the fourth transmembrane segment in each domain (the S4 segment) and is likely to be involved in voltage-gating. The positively charged side chains on the S4 segment are likely to be paired with the negatively charged side chains on the other five segments such that membrane depolarization could shift the position of one helix relative to the other, thereby opening the channel. Accessory subunits may modify the function of the channel.

Therapeutic utility in recombinant materials derived from the DNA of the numerous sodium channels have been discovered. For example, U.S. Patent No. 5,132,296 by Cherksey discloses purified Na+ channels that have proven useful as therapeutic and diagnostic tools.

Isoforms of sodium channels are divided into ..subfamilies. The term Jsoform is used to mean distinct but closely related sodium channel proteins, i.e., those having an amino acid homology of approximately 60-80%. These also show strong homology in functions.

The term ..subfamilies is used to mean distinct sodium channels that have an amino acid homology of approximately 80-95%. Combinations of several factors are used to determine the distinctions within a subfamily, for example, the speed of a channel, chromosomal location, expression data, homology to other channels within a species, and homology to a channel of the same subfamily across species. Another consideration is an affinity to tetrodotoxin („TTX“). TTX is a highly potent toxin from the puffer or fugu fish which blocks the conduction of nerve impulses along axons and in excitable membranes of nerve fibers.

TTX binds to the Na* channel and blocks the flow of sodium ions.

Studies employing TTX as a probe have shed much light on the mechanism and structure of Na* channels. There are three Na* channel subtypes that are defined by the affinity for TTX, which can be measured by the IC50 values: TTX-sensitive Na* channels (IC50 = 1-30 nM), TTX-insensitive Na* channels (IC50 - 1-5 μΜ), and TTX-resistant Na* channels (IC50 2: 50 μΜ).

TTX-insensitive action potentials were first studied in rat skeletal muscle (Redfern et al., Acta Physiol. Scand. 82,70-78 (1971)). Subsequently, these action potentials were described in other mammalian tissues, including newborn mammalian skeletal muscle, mammalian cardiac muscle, mouse dorsal root ganglion cells in vitro and in culture, cultured mammalian skeletal muscle and L6 cells. See Rogart, Ann. Rev. Physiol. 43, 711-725 (1980).

Rat dorsal root ganglia neurons possess both TTX-sensitive (IC50 ~ 0.3 nM) and TTXresistant (IC50 - 100 μΜ) sodium channel currents, as described in Roy et al., J. Neurosci. 12, 2104-2111 (1992). TTX-resistant sodium currents have also been measured in rat nodose and petrosal ganglia. See Ikeda et al., J. Neurophysiol. 55, 527-539 (1986) and Stea et al., Neurosci. 47, 727-736 (1992). Electrophysiologists believe that another TTX-resistant sodium channel is yet to be detected.

Though cDNAs from rat skeletal muscle, heart and brain are known, identification and isolation of cDNA from peripheral sensory nerve tissue, such as dorsal root ganglia, has been hampered by the difficulty of working with such tissue.

. SUMMARY OF THE INVENTION The present invention provides novel purified and isolated nucleic acid sequences encoding mammalian, preferably TTX-resistant, nervous tissue sodium channel proteins that are strongly expressed in adult DRG and nodose ganglia, less strongly expressed in brain, spinal cord and superior cervical ganglia, and not expressed in sciatic nerve, heart or skeletal muscle. In presently preferred forms, novel DNA sequences comprise cDNA sequences encoding rat nervous tissue sodium channel protein. One aspect of the present invention is the α-subunit of this sodium channel protein.

Disclosed is the DNA, cDNA, and mRNA derived from the nucleic acid sequences of the invention and the cRNA derived from the mRNA. Specifically, two cDNA sequences together encode for the full length rat nervous tissue sodium channel.

Also included in this invention are alternate DNA forms, such as genomic DNA, DNA prepared by partial or total chemical synthesis from nucleotides, and DNA having deletions or mutations.

Still another aspect of the invention is the novel rat TTX-resistant sodium channel protein and fragments thereof, encoded by the DNA of this invention.

Another aspect of the present invention are recombinant polynucleotides and oligonucleotides comprising a nucleic acid sequence derived from the DNA sequence of this invention.

Another aspect of the invention is a method of stabilizing the full length cDNA which encodes the protein sequence of the invention.

Further aspects of the invention include expression vectors comprising the DNA of the invention, host cells transformed or transfected by these vectors, and a cDNA library of these host cells.

Also forming part of this invention is an assay for inhibitors of the sodium channel protein comprising contacting a compound suspected of being an inhibitor with expressed sodium channel and measuring the activity of the sodium channel.

Further provided is a method of inhibiting the activity of the TTX-resistant sodium channel comprising administering an effective amount of a compound having an IC50 of 10 μΜ or less.

Additionally provided are methods of employing the DNA for forming monoclonal and polyclonal antibodies, for use as molecular targets for drug discovery, highly specific markers for specific antigens, detector molecules, diagnostic assays, and therapeutic uses, such as pain relief, a probe for the PN5 channel in other mammalian tissue, designing therapeutics and screening for therapies.

BRIEF DESCRIPTION OF THE SEP ID'S AND FIGURES Figures 1A-E depict the 5908 nucleotide cDNA native sequence encoding the rat sodium channel type 5 („PN5“) (SEQ ID NO: 1), derived from two overlapping cDNA clones, designated 26.2 and 1.18.

Figures 2A-F depict the deduced amino acid sequence of PN5 (SEQ ED NO: 2, represented in the three-letter amino acid code). Figures 2G-H, depicting the deduced amino acid sequence of PN5 in single letter amino acid code, also show the homologous domains (IIV); the putative transmembrane segments (S1-S6); the amino acid conferring resistance to TTX (♦); N-glycosylation sites (·); cAMP-dependent protein kinase A (PKA) phosphorylation site (0); and the termination codon (*).

Figure 3A depicts an 856 base pair sequence for the human PN5 (SEQ ID NO: 3). Figure 3B depicts the amino acid sequence comparison of the hPN5 fragment with rat PN5.

Figure 4 depicts the sequence for the novel sodium channel domain IV probe (SEQ ED NO: 4).

Figures 5A-E depict the 5334 nucleotide sequence modified for stability and expression (SEQ ID NO: 5). Nucleotides 24 to 5518 constitute the 5295 bp region coding for a 1765 amino acid protein.

Figure 6 depicts the cloning map of PN5.

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a purified and isolated nucleic acid sequence encoding for a novel mammalian, preferably TTX-resistant, sodium channel protein. The term purified and isolated DNA refers to DNA that is essentially free, i.e. contains less than about 30%, preferably less than about 10%, and even more preferably less than about 1%, of the DNA with which the DNA of interest is naturally associated. Techniques for assessing purity are well known to the art and include, for example, restriction mapping, agarose gel electrophoresis, and CsCl gradient centrifugation.

The term DNA is meant to include „cDNA“, or complementary DNA, which is single-stranded or double-stranded DNA sequences made by reverse transcription of mRNA isolated from a donor cell or by chemical synthesis. For example, treatment of mRNA with a reverse transcriptase such as AMV reverse transcriptase or M-MuLV reverse transcriptase in the presence of an oligonucleotide primer will furnish an RNA-DNA duplex which can be treated with RNase H, DNA polymerase, and DNA ligase to generate double-stranded cDNA. If desired, the double-stranded cDNA can be denatured by conventional techniques such as heating to generate single-stranded cDNA. The term „cDNA“ includes cDNA that is a complementary copy of the naturally occurring mRNA ,as well as complementary copies of variants of the naturally occurring mRNA that have the same biological activity. Variants would include, for example, insertions, deletions, sequences with degenerate codons and alleles. „cRNA“ corresponding to mRNA transcribed from a DNA sequence encoding the asubunit of a novel, preferably TTX-resistant, sodium channel protein is contemplated by this invention. The term „cRNA“ refers to RNA that is a copy of the mRNA transcribed by a cell.

Specifically, the invention encompasses DNA having the native versions of the nucleotide sequences set forth in Figures 1A-E (SEQ ID NO: 1) designated herein as sodium channel type 5 (PN5). Figures 1A-E depict the 5908 nucleotide cDNA construct comprising a 5298-base (counting the stop codon) open reading frame (SEQ ID NO:1). Nucleotide residue 79 represents the start site of translation and residue 5376 represents the end of the stop codon.

The invention also encompasses engineered versions of PN5, and specifically the version as set forth in Figures 5A-E (SEQ ID NO: 5). This 5334 nucleotide SaH-Xbal clone lacks most of the untranslated sequences, the 5298 nucleotide open reading frame beginning at nucleotide 24 and ending at nucleotide 5321. The start and stop codons are underlined, as are the translationally silent mutations at nucleotides 3932, 3935, 3941, 3944, and 3947, which were introduced to block rearrangement in this region during growth in £. Coli.

The nucleotide sequence of SEQ ED NO: 1 (Figures 1A-E) corresponds to the cDNAs from rat. A homology search provided that the closest related sodium channel is found in the rat cardiac channel, with 72.5% homology. The next closely related channels are rPNl, with 72% and rat brain types I and ΕΠ, with 71.8% and 71.3% respectively. Homology to rPN3a, hPN3, rPN4, rPN4a, rat brain type Π and rat skeletal muscle are each approximately 70 to 71%.

Additionally, an 856 base pair clone (SEQ ED NO: 3) as shown in Figure 3A has been isolated from a human dorsal root ganglia (DRG) „cDNA library*1 and is closely related to the rat PN5 amino acid sequence with 79% identity and 86% homology. The human PN5 sequence spans the region between IUSl and interdomain ΕΠ/IV which includes the fast inactivation gate (i.e., IFM) that is located within interdomain ΠΙ/IV.

The term „cDNA library*' refers to a collection of clones, usually in a bacteriophage, or less commonly in bacterial plasmids, containing cDNA copies of mRNA sequences derived from a donor cell or tissue.

It is believed that additional homologs of the novel rat TTX-resistant sodium channel described herein are also expressed in other mammalian tissue.

Northern blot analysis (Example 5) indicates that PN5 is encoded by a -6.5 kb ........ . transcript.

The deduced amino acid sequence of PN5, shown in Figures 2A-F (SEQ ED NO: 2), exhibits the primary structural features of an α-subunit of a voltage-gated, TTX-resistant sodium channel. Shown in Figures 2G-H are the homologous domains (I-IV); the putative transmembrane segments (S1-S6); the amino acid conferring resistance to TTX (♦); Nglycosylation sites (·); and cAMP-dependent PKA phosphorylation sites (0). DNA sequences encoding the same or allelic variant or analog sodium channel protein polypeptides of the nervous system, through use of, at least in part, degenerate codons are also contemplated by this invention.

An interesting feature of this deduced amino acid sequence is that the amino acid that is most responsible for TTX-sensitivity is located at position 355 and is not aromatic. In rat and human brain type sodium channels, skeletal muscle channel, and in PNl and PN4, this amino acid is tyrosine or phenylalanine and these channels are all TTX-sensitive. In PN3 and PN5, the amino acid is a serine. Since PN3 is highly resistant to TTX, the implication is that PN5 is also a TTX-resistant channel. The cardiac channel has a cysteine at this position and is ,jnsensitive“ to TTX.

Although PN5 contains all of the hallmark features of a voltage-gated sodium channel, it has unique structural features that distinguish it from other sodium channels. For example, DHS4 has 5 basic amino acids conserved in all sodium channels that could play a significant role in the voltage sensing aspects of the channel function. In PN5, the first basic amino acid is replaced by an alanine. Similarly, in DIHS4, PN5 has 5 basic amino acids rather than six that are present in other sodium channel sequences, the last arginine replaced by a glutamine. In DIIIS3, the transmembrane segment contains only 18 amino acids, in contrast to 22 amino acids in other channels. Also, the short linker (4 amino acids) loop between S3 and S4 in DIH is even shorter by a .deletion’ of 3 amino acids. This shortening of the S3 and the linker loop has been confirmed by designing primers in the appropriate region of the sequence for an RTPCR experiment from rat DRG and sequencing the amplified DNA fragment. Such an experiment has been performed to confirm the sequence of another region of PN5, in the DIVS5-S6 loop, where there was a deletion of an 8 amino acid peptide.

Reverse transcription-polymerase chain reaction (oligonucleotide-primed RT-PCR) tissue distribution analysis of RNA from the rat central and peripheral nervous systems, in particular from rat DRG, was performed. Eight main tissue types were screened for expression of the unique PN5 genes corresponding to positions 5651-5903 of SEQ ID NO: 1 (Figures 1A-E). PN5 mRNA was present in five of the tissues studied: brain, spinal cord, DRG, nodose ganglia, and superior cervical ganglia. PN5 was not present in the remaining tissues studied: sciatic nerve tissue, heart or skeletal muscle tissue. PN5 was found to be the strongest in DRG and nodose ganglia, leading the applicants to believe that the DRG is enriched with PN5. PN5 shows dramatic abundance differences across a range of tissues.

PN5 has a gradient of expression with high expression in DRG. PN5 has a gradient of expression like other channels, but more limited distribution.

The invention not only includes the entire protein expressed by the cDNA sequences of SEQ ID NOS: 1, 2 and 3, but also includes protein fragments. These fragments can be obtained by cleaving the full length proteins or by using smaller DNA sequences or ..polynucleotides to express the desired fragment.

The term polynucleotide as used herein refers to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. This tenn refers only to the primary structure of the molecule. Thus, this term includes double- and single-stranded DNA, as well as double- and single-stranded RNA. It also includes modified, for example, by methylation and/or by capping, and unmodified forms of the polynucleotide.

Further, the term polynucleotide is intended to include a recombinant polynucleotide, which is of genomic, cDNA, semisynthetic or synthetic origin which, by virtue of its origin or manipulation is not associated with all or a portion of the polynucleotide with which it is associated in nature and/or is linked to a polynucleotide other than that to which it is linked in nature. ...........

Accordingly, the invention also includes polynucleotides that can be used to make polypeptides of about 10 to 1500, preferably 10 to 100, amino acids in length. The isolation and purification of such recombinant polypeptides can be accomplished by techniques that are well known in the art, for example, preparative chromatographic separations or affinity chromatography. In addition, polypeptides can also be made by synthetic means which are well known in the art.

The invention allows for the manipulation of genetic materials by recombinant technology to produce polypeptides that possess the structural and functional characteristics of the novel voltage-gated, TTX-resistant sodium channel α-subunit found in sensory nerves.

Site directed mutagenesis can be used to provide such recombinant polypeptides. For example, synthetic oligonucleotides can be specifically inserted or substituted into the portion of the gene of interest to produce genes encoding for and expressing a specific mutant. Random degenerate oligonucleotides can also be inserted and phage display techniques can be used to identify and isolate polypeptides possessing a functional property of interest.

In addition, the present invention contemplates recombinant polynucleotides of about 15 to 20kb, preferably 10 to 15kb, nucleotides in length, comprising a nucleic acid sequence „derived from the DNA of the invention.

The term derived from a designated sequence, refers to a nucleic acid sequence that is comprised of a sequence of approximately at least 6 to 8 nucleotides, more preferably at least 10 to 12 nucleotides, and, even more preferably, at least 15 to 20 nucleotides that correspond to, i.e., are homologous or complementary to, a region of the designated sequence. The derived sequence is not necessarily physically derived from the nucleotide sequence shown, but may be derived in any manner, including for example, chemical synthesis or DNA replication or reverse transcription, which are based on the information provided by the sequences of bases in the region(s) from which the polynucleotide is derived.

A neonatal expression test was performed with FI 1, a fusion cell line designed from neonatal rat DRG fused with a mouse cell line, N18TG, from Massachusetts General Hospital, FI 1 responds to trophic agents, such as NGF, by extending dendrites. It was found that PN5 was present in both native FI 1 and Fl 1 treated with NGF, leading the applicants to believe that the sodium channel is natively expressed in Fl 1.

In situ hybridization of PN5 mRNA to rat DRG tissue provides localization predominantly in the small and medium neurons with no detection in large neurons.

PN5 was also mapped to its cytogenetic location on mouse chromosome preparations.

PN5 maps to the same chromosome as the cardiac channel and PN3.

In general, sodium channels comprise an a- and two B-subunits. The B-subunits may modulate the function of the channel. However, since the α-subunit is all that is required for the channel to be fully functional, expression of the cDNA in SEQ ID NO: 1 (Figures 1A-E) will provide a fully functional protein. The gene encoding the Brsubunit in peripheral nerve tissue was found to be identical to that found in rat heart, brain and skeletal muscle. The cDNA of the B,-subunit is not described herein as it is well known in the art, see Isom et al., Neuron 12,1183-1194 (1994). However, it is to be understood that by combining the known sequence for the Bj-subunit with the α-subunit sequence described herein, one may obtain complete PN5 voltage-gated, preferably TTX-resistant, sodium channel.

The present invention also includes ..expression vectors comprising the DNA or the cDNA described above, host cells transformed with these expression vectors capable of producing the sodium channel of the invention, and cDNA libraries comprising such host cells.

The term expression vector refers to any genetic element, e.g., a plasmid, a chromosome, a virus, behaving either as an autonomous unit of polynucleotide expression within a cell or being rendered capable of replication by insertion into a host cell chromosome, having attached to it another polynucleotide segment, so as to bring about the replication and/or expression of the attached segment. Suitable vectors include, but are not limited to, plasmids, bacteriophages, and cosmids. Vectors will contain polynucleotide sequences which are necessary to effect ligation or insertion of the vector into a desired host cell and to effect the expression of the attached segment. Such sequences differ depending on the host organism, and will include promoter sequences to effect transcription, enhancer sequences to increase transcription, ribosomal binding site sequences and transcription and translation termination sequences.

The term host cell generally refers to prokaryotic or eukaryotic organisms and includes any transformable or transfectable organism which is capable of expressing a protein and can be, or has been, used as a recipient for expression vectors or other transferred DNA. Host cells can also be made to express protein by direct injection with exogenous cRNA translatable into the protein of interest. A preferred host cell is the Xenopus oocyte.

The term transformed refers to any known method for the insertion of foreign DNA or RNA sequences into a host prokaryotic cell. The term ..transfected refers to any known method for the insertion of foreign DNA or RNA sequences into a host eukaryotic cell. Such transformed or transfected cells include stably transformed or transfected cells in which the inserted DNA is rendered capable of replication in the host cell. They also include transiently expressing cells which express the inserted DNA or RNA for limited periods of time. The transformation or transfection procedure depends on the host cell being transformed. It can include packaging the polynucleotide in a virus as well as direct uptake of the polynucleotide, such as, for example, lipofection or microinjection. Transformation and transfection can result in incorporation of the inserted DNA into the genome of the host cell or the maintenance of the inserted DNA within the host cell in plasmid form. Methods of transformation are well known in the art and include, but are not limited to, viral infection, electroporation, lipofection, and calcium phosphate mediated direct uptake.

It is to be understood that this invention is intended to include other forms of expression vectors, host cells, and transformation techniques which serve equivalent functions and which become known to the art hereto.

The invention also pertains to an assay for inhibitors of the novel TTX-resistant sodium channel protein comprising contacting a compound suspected of being an inhibitor with expressed sodium channel and measuring the activity of the sodium channel. The compound can be a substantially pure compound of synthetic origin combined in an aqueous medium, or the compound can be a naturally occurring material such that the assay medium is an extract of biological origin, such as, for example, a plant, animal, or microbial cell extract.

PN5 activity can be measured by methods such as electrophysiology (two electrode voltage clamp or single electrode whole cell patch clamp), guanidinium ion flux assays, and toxinbinding assays. An inhibitor is defined as generally that amount that results in greater than 50% decrease in PN5 activity, preferably greater than 70% decrease in PN5 activity, more preferably greater than 90% decrease in PN5 activity.

Many uses of the invention exist, a few of which are described below: 1. Probe for mamalian channels.

As mentioned above, it is believed that additional homologs of the novel rat TTXresistant sodium channel described herein are also expressed in mammalian tissue, in particular, human tissue. The entire cDNAs of PN5 rat sodium channels of the present invention can be used as a probe to discover whether additional novel PN5 voltage-gated, preferably TTX-resistant, sodium channels exist in human tissue and, if they do, to aid in isolating the cDNAs for the human protein.

The human homologues of the rat TTX-resistant PN5 channels can be cloned using a human DRG cDNA library. Human DRG are obtained at autopsy. The frozen tissue is homogenized and the RNA extracted with guanidine isothiocyanate (Chirgwin et al. Biochemisny 18, 5294-5299, (1979)). The RNA is size-fractionated on a sucrose gradient to enrich for large mRNAs because the sodium channel oc-subunits are encoded by large (7-11 kb) transcripts. Double-stranded cDNA is prepared using the Superscript Choice cDNA kit (GIBCO BRL) with either oligo(dT) or random hexamer primers. EcoRI adapters are ligated onto the double-stranded cDNA which is then phosphorylated. The cDNA library is constructed by ligating the double-stranded cDNA into the bacteriophage-lambda ZAP Π vector (Stratagene) followed by packaging into phage particles.

Phage are plated out on 150 mm plates on a lawn of XLI-Blue MRP bacteria (Stratagene) and plaque replicas are made on Hybond N nylon membranes (Amersham).

Filters are hybridized to rat PN5 cDNA probes by standard procedures and detected by autoradiography or chemiluminescence. The signal produced by the rat PN5 probes hybridizing to positive human clones at high stringency should be stronger than obtained with rat brain sodium channel probes hybridizing to these clones. Positive plaques are further purified by limiting dilution and re-screened by hybridization or PCR. Restriction mapping and polymerase chain reaction will identify overlapping clones that can be assembled by standard techniques into the full-length human homologue of rat PN5. The human clone can be expressed by injecting cRNA transcribed in vitro from the full-length cDNA clone into Xenopus oocytes, or by transfecting a mammalian cell line with a vector containing the cDNA linked to a suitable promoter. 2. Antibodies Against PN5.

The polypeptides of the invention are highly useful for the development of antibodies against PN5. Such antibodies can be used in affinity chromatography to purify recombinant sodium channel proteins or polypeptides, or they can be used as a research tool. For example, antibodies bound to a reporter molecule can be used in histochemical staining techniques to identify other tissues and cell types where PN5 are present, or they can be used to identify epitopic or functional regions of the sodium channel protein of the invention.

The antibodies can be monoclonal or polyclonal and can be prepared by techniques that are well known in the art. Polyclonal antibodies are prepared as follows: an immunogenic conjugate comprising PN5 or a fragment thereof, optionally linked to a carrier protein, is used to immunize a selected mammal such as a mouse, rabbit, goat, etc. Serum from the immunized mammal is collected and treated according to known procedures to separate the immunoglobulin fraction.

Monoclonal antibodies are prepared by standard hybridoma cell technology based on that reported by Kohler and Miistein in Nature 256,495-497 (1975). Spleen cells are obtained from a host animal immunized with the PN5 protein or a fragment thereof, optionally linked to a earner. Hybrid cells are formed by fusing these spleen cells with an appropriate myeloma cell line and cultured. The antibodies produced by the hybrid cells are screened for their ability to bind to expressed PN5 proteins.

A number of screening techniques well known in the art, such as, for example, forward or reverse enzyme-linked immunosorbent assay screening methods, may be employed. The hybrid cells producing such antibodies are then subjected to recloning and high dilution conditions in order to select a hybrid cell that secretes a homogeneous population of antibodies specific to either the PN5 protein.

In addition, antibodies can be raised by cloning and expressing nucleotide sequences or mutagenized versions thereof coding at least for the amino acid sequences required for specific binding of natural antibodies, and these expressed proteins used as the immunogen.

Antibodies may include the complete immunoglobulin or a fragment thereof. Antibodies may be linked to a reporter group such as is described above with reference to polynucleotides.

Example 10 illustrates practice of producing an antibody. 3. Therapeutic Targets for Compounds to Treat Disorders and Assays Thereof.

The present invention also includes the use of the novel voltage-gated, preferably TTXresistant, sodium channel α-subunit as a therapeutic target for compounds to treat disorders of the nervous system based on the RT-PCR localization data. The disorders include, but are not limited to, epilepsy, stroke injury, brain injury, diabetic neuropathy, traumatic injury, chronic neuropathic pain, and AIDS-associated neuropathy. 4. Designing Therapeutics based on Inhibiting PN5 and assays thereof.

This invention is also directed to inhibiting the activity of PN5 in brain, spinal cord, DRG, nodose ganglia, and superior cervical ganglia tissues. However, it is to be understood that further studies may reveal that PN5 is present in other tissues, and as such, those tissues can also be targeted areas. For example, the detection of PN5 mRNA in nodose ganglia suggests that PN5 may conduct TTX-resistant sodium currents in this and other sensory ganglia of the nervous system.

In addition, it has been found that proteins not normally expressed in certain tissues are expressed in a disease state. Therefore, this invention is intended to encompass the inhibition of PN5 in tissues and cell types where the protein is normally expressed, and in those tissues and cell types where the protein is only expressed during a disease state.

For example, it is believed that TTX-resistant sodium channels play a key role in transmitting nerve impulses relating to sensory inputs such as pain and pressure. This information will facilitate the design of therapeutics that can be targeted to a specific area such as peripheral nerve tissue.

The recombinant protein of the present invention can be used to screen for potential therapeutics that have the ability to inhibit the sodium channel of interest In particular, it would be useful to inhibit selectively the function of sodium channels in peripheral nerve tissues responsible for transmitting pain and pressure signals without simultaneously affecting the function of sodium channels in other tissues such as heart and muscle. Such selectivity would allow for the treatment of pain without causing side effects due to cardiac or neuromuscular complications. Therefore, it would be useful to have DNA sequences coding for sodium channels that are selectively expressed in peripheral nerve tissue.

. Pain Reliever.

Sodium channels in peripheral nerve tissue play a large role in the transmission of nerve impulses, and therefore are instrumental in understanding neuropathic pain transmission. Neuropathic pain falls into two components: allodynia, where a normally non-painful stimulus becomes painful, and hyperalgesia, where a usually normal painful stimulus becomes . .. .. extremely painful.

In tissue localization studies, PN5 mRNA maps small and medium neurons of DRG. PN5 mRNA is also present in brain and spinal cord. Inhibiting its activities may help prevent ailments such as headaches and migraines. The ability to inhibit the activity of these sodium channels, i.e., reduce the conduction of nerve impulses, will affect the nerve’s ability to transmit pain impulses. Selective inhibition of sodium channels in sensory neurons such as DRG will allow the blockage of pain impulses without complicating side effects caused by inhibition of sodium channels in other tissues such as brain and heart. In addition, certain diseases are caused by sodium channels that produce impulses at an extremely high frequency. The ability to reduce the activity of the channel can then eliminate or alleviate the disease. Accordingly, potential therapeutic compounds can be screened by methods well known in the art to discover whether they can inhibit the activity of the recombinant sodium channel of the invention. Barram, M. et al., Naun-Schmiedeberg’s Archives of Pharmacology 347, 125-132 (1993) and McNeal, E.T. et al., J. Med. Chem. 28, 381-388 (1985). For similar studies with the acetyl choline receptor, see, Claudio et al., Science 238, 1688-1694 (1987).

For example, pain can be alleviated by inhibiting the activity of the novel preferably TTX-resistant sodium channel comprising administering a therapeutically effective amount of a compound having an IC50 approximately 10 μΜ or less, preferably <1 μΜ. Potential therapeutic compounds are identified based on their ability to inhibit the activity of PN5. Therefore, the aforementioned assay can be used to identify compounds having a therapeutically effective IC50· The term „IC50“ refers to the concentration of a compound that is required to inhibit by 50% the activity of expressed PN5 when activity is measured by electrophysiology, flux assays, and toxin-binding assays, as mentioned above. 6. Diagnostic Assays.

The basic molecular biology techniques employed in accomplishing features of this invention, such as RNA, DNA and plasmid isolation, restriction enzyme digestion, preparation and probing of a cDNA library, sequencing clones, constructing expression vectors, transforming cells, maintaining and growing cell cultures, and other general techniques are well known in the art, and descriptions of such techniques can be found in general laboratory manuals such as Molecular Cloning: A Laboratory Manual by Sambrook et al. (Cold Spring Harbor Laboratory Press, 2nd edition, 1989).

For example, the polynucleotides of the invention can be bound to a ..reporter molecule to form a polynucleotide probe useful for Northern and Southern blot analysis and in situ hybridizations.

The term reporter molecule refers to a chemical entity capable of being detected by a suitable detection means, including, but not limited to, spectrophotometric, chemiluminescent, immunochemical, or radiochemical means. The polynucleotides of this invention can be conjugated to a reporter molecule by techniques well known in the art. Typically the reporter molecule contains a functional group suitable for attachment to or incorporation into the polynucleotide. The functional groups suitable for attaching the reporter group are usually activated esters or alkylating agents. Details of techniques for attaching reporter groups are well known in the art. See, for example, Matthews, J.A., Batki, A., Hynds, C., and Kricka, L.J., Anal. .Biochem. 151, 205-209 (1985) and Engelhardt et al., European Patent Application No. 0302175.

Accordingly, the following Examples are merely illustrative of the techniques by which the invention can be practiced.

Abbreviations The following abbreviations are used throughout the Examples and have each of the respective meanings defined below.

BSA: bovine serum albumin Denhardt’s solution: 0.02% BSA, 0.02% polyvinyl-pyrrolidone, 0.02% Ficoll (0.1 g BSA, 0.1 g Ficoll and 0.1 g polyvinylpyrrolidone per 500 ml) DRG: dorsal root ganglia EDTA: Ethylenediaminetetraacetic acid, tetrasodium salt MEN: 20 mM MOPS, 1 mM EDTA, 5 mM sodium acetate, pH 7.0 MOPS: 3-(N-morpholino)propanesulfonic. acid (Sigma Chemical Company) PN5: peripheral nerve sodium channel 5 PNS: peripheral nervous system SDS: sodium dodecyl sulfate SSC: 150 mM NaCl, 15 mM sodium citrate, pH 7.0 SSPE: 80 mM NaCl, 10 mM sodium phosphate, 1 mM ethylenediaminetetraacetate, pH 8.0 TEV: two electrode voltage clamp TTX: tetrodotoxin (Sigma Chemical Company) EXAMPLES The following Examples illustrate practice of the invention.

Materials The plasmid pBK-CMV was obtained from Stratagene (La Jolla, CA); the plasmid 5 pBSTA is described by Goldin et al., in Methods in Enzymology (Rudy & Iverson, eds.) 207, 279-297; the plasmid pCIneo was obtained from Promega (Madison, WI); and the plasmid pCRB was obtained from Invitrogen (Carlsbad, CA).

The oocyte expression vector plasmid pBSTAcIIr was constructed from pBSTA by insertion of a synthetic oligonucleotide linker; plasmid pKK232-8 was obtained from Pharmacia Biotech (Piscataway, NJ); plasmid pCRU was obtained from Invitrogen, San Diego, CA. Competent E. coli cell lines STBL2™ and SURE® were obtained from Gibco/BRL and Stratagene, respectively.

EXAMPLE 1 OBTAINING RNA FROM RAT DRG, BRAIN AND SPINAL CORD Lumbar DRG No. 4 and No. 5 (L4 and L5) brain and spinal cord were removed from anesthetized adult male Sprague-Dawley rats under a dissecting microscope. The tissues were frozen in dry ice and homogenized with a Polytron homogenizer; the RNA was extracted by the guanidine isothiocyanate procedure (see Chomczynksi et al., Anal. Biochemistry 162,15620 159 (1987)). Total RNA (5 pg of each sample) was dissolved in MEN buffer containing 50% formamide, 6.6% formaldehyde and denatured at 65°C for 5-10 min. The RNA was electrophoresed through a 0.8% agarose gel containing 8.3% formaldehyde in MEN buffer. The electrode buffer was MEN buffer containing 3.7% formaldehyde; the gel was run at 50 V for 12-18 hours.

Size markers, including ribosomal 18S and 28S RNAs and RNA markers (GIBCO BRL), were run in parallel lanes of the gel. Their positions were determined by staining the excised lane with ethidium bromide (0.5 pg/ml) followed by photography under UV light.

After electrophoresis, the gel was rinsed in 2xSSC and the RNA was transferred to a Duralose membrane (Stratagene) with 20xSSC by capillary action; the membrane was baked under vacuum at 80°C for 1 hour.

EXAMPLE 2 PROBE FROM RAT BRAIN HA A 32P-labeled cRNA probe complementary to nucleotides 4637-5868 of the rat brain HA sodium channel ot-subunit sequence was synthesized in vitro with T7 RNA polymerase (Pharmacia) using pEAF8 template DNA, (Noda et al., Nature 320,188-192 (1986)) that had been linearized with BstEH.

Protocols for each procedure mentioned above can be found in Molecular Cloning; A Laboratory Manual by Sambrook et al. (Cold Spring Harbor Laboratory Press, 2nd edition, 1989).

EXAMPLE 3 HYBRIDIZATION OF RNA WITH THE PROBE FROM RAT BRAIN HA The membrane of Example 1 was prehybridized in 50% formamide, 5xSSC, 50 mM 20 sodium phosphate, pH 7.1, lx Denhardt’s solution, 0.5% SDS, and sheared, heat-denatured salmon sperm DNA (1 mg/ml) for 16 hours at 42°C. The membrane was hybridized in 50% formamide, 5xSSC, 50 mM sodium phosphate, pH 7.1, lx Denhardt’s solution, 0.5% SDS, and sheared, heat-denatured salmon sperm DNA (200 gg/ml) with the 32P-labeled cRNA probe (ca. l-3xl06 cpm/ml) described in Example 2 for 18 hours at 42°C.

The membrane was rinsed with 2xSSC, 0.1% SDS at room temperature for 20 min. and then washed sequentially with: 2xSSC, 0.1%- SDS at 55°C for 30 min., 0.2xSSC, 0.1% SDS at 65°C for 30 min., 0.2xSSC, 0.1% SDS at 70°C for 30 min., and 0.2xSSC, 0.1% SDS, 0.1% sodium pyrophosphate at 70°C for 20 min. The filter was exposed against Kodak X-omat AR film at -80°C with intensifying screens for up to 2 weeks.

The pEAF8 probe hybridized to mRNAs in the DRG sample with sizes of 11 kb, 9.5 kb, 7.3 kb, and 6.5 kb, estimated on the basis of their positions relative to the standards.

EXAMPLE 4 NOVEL SODIUM CHANNEL DOMAIN TV PROBE The probe was obtained as follows: RT-PCR was performed on RNA isolated from rat DRG using degenerate oligonucleotide primers that were designed based on the homologies between known sodium channels in domain IV. The domain TV products were cloned into a plasmid vector, transformed into E. coli and single colonies isolated. The domain IV specific PCR products obtained from several of these colonies were individually sequenced. Cloned novel domain IV sequence was as follows (SEQ ID NO: 4): CTCAACATGG TTACGATGAT GGTGGAGACC GACGAGCAGG GCGAGGAGAA GACGAAGGTT CTGGGCAGAA TCAACCAGTT CTTTGTGGCC GTCTTCACGG 101 GCGAGTGTGT GATGAAGATG TTCGCCCTGC GACAGTACTA TTTCACCAAC 151 GGCTGGAACG TGTTCGACTT CATAGTGGTG ATCCTGTCCA TTGGGAGTCT 201 GCTGTTTCT GCAATCCTTA AGTCACTGGA AAACTACTTC TCCCCGACGC 251 TCTTCCGGGT CATCCGTCTG GCCAGGATCG GCCGCATCCT CAGGCTGATC 301 CGAGCAGCCA AGGGGATTCG CACGCTGCTC TTCGCCCTCA TGATGTCCCT 351 GCCCGCCCTC TTCAACATCG GCCTCCTCCT CTTCCTCGTC ATGTTCATCT 401 ACTCCATCTT CGGCATGGCC AGCTTCGCTA ACGTCGTGGA CGAGGCCGGC 451 ATCGACGACA TGTTCAACTT CAAGACCTTT GGCAACAGCA TGCTGTGCCT 501 GTTCCAGATC ACCACCTCGG CCGGCTGGGA CGGCCTCCTC AGCCCCATCC 551 TCAACACGGG GCCTCCCTAC TGCGACCCCA ACCTGCCCAA CAGCAACGGC 601 TCCCGGGGGA ACTGCGGGAG CCCGGCGGTG GGCATCATCT TCTTCACCAC 651 CTACATCATC ATCTCCTTCC TCATCGTGGT CAACATGTAT ATCGCAGTCA 701 TC This sequence was labeled with 32P by random priming.

EXAMPLE 5 HYBRIDIZATION OF RNA WITH THE NOVEL SODIUM CHANNEL 3’-UTR PROBE A Northern blot was prepared with lOpg total RNA from rat brain, spinal cord, and DRG. The blot was hybridized with a cRNA probe from the 3‘-UTR. The 3‘-UTR was cloned into pSP 73 vector, the cRNA transcribed using a Trans Probe T kit (Pharmacia Biotech) and 32P UTP. The blot was prehybridized for 2 hours at 65°C in a solution containing 5XSSC, IX Denhardt's solution, 0.5% SDS, 50mM sodium phosphate, pH 7.1, salmon sperm DNA (lmg/ml) and 50% formamide. Hybridization was conducted at 45°C for 18 hours in the above solution except that the salmon sperm DNA was included at a concentration of 200gg/ml and the ^^P-labeled probe was added at 7.5x10^ cpm.ml solution. The blot was subsequently washed three times at 2XSSC and 0.1% SDS at room temperature, once with 0.2XSSC and 0.1% SDS at 65°C for 20 min., and once with 0.2XSSC, 0.1% SDS and 0.1% sodium pyrophosphate at 65°C for 20 min. The blot was analyzed on a Phospholmager (BioRad) after an exposure of 2 days. The results indicated that there was a ~6.5kb band signal present in brain only in the lane containing RNA from DRG. Because of the lower abundance of PN5 mRNA, as evidenced by the RT-PCR experiment, the 6.5kb band was not detectable in brain and spinal cord.

EXAMPLE 6 CONSTRUCTION & SCREENING OF cDNA LIBRARY FROM RAT DRG An EcoRI-adapted cDNA library was prepared from normal adult male SpragueDawley rat DRG poly(A)+ RNA using the Superscript Choice System (GIBCO BRL). cDNA (>4 kb) was selected by sucrose gradient fractionation as described by Kieffer, Gene 109, 115119 (1991). The cDNA was then ligated into the Zap Express vector (Stratagene), and packaged with the Gigapack H XL lambda packaging extract (Stratagene). Similarly, a >2kb DRG cDNA library was synthesized.

Phage (3.5x10s) were screened by filter hybridization with a 32P-labeled probe (rBIIa, bases 4637-5868 as follows of Auld el at, Neuron 1, 449-461 (1988)). Filters were hybridized in 50% formamide, 5X SSPE, 5X Denhardt’s solution, 0.5% SDS, 250pg/ml sheared, denatured salmon sperm DNA, and 50 mM sodium phosphate at 42°C and washed in 0.5X SSC/0.1% SDS at 50°C.

Southern blots of EcoRI-digested plasmids were hybridized with the 32P-labeled DNA probe, (SEQ ID NO: 4). The filters were then hybridized in 50% formamide, 6X SSC, 5X Denhardt’s solution, 0.5%, SDS, and 100pg/ml sheared, denatured salmon sperm DNA at 42°C and were washed in 0.1X SSC/0.1% SDS at 656C.

Positive clones were excised in vivo into pBK-CMV using the ExAssist/XLOLR system (Stratagene).

EXAMPLE 7 CLONES AND NUCLEOTIDE ANALYSIS cDNA clones, 26.2 and 25.1 were isolated from the >4kb DRG cDNA library and clone 1.18 was isolated from the >2kb DRG cDNA library. By sequence analysis, 26.2 appeared to be a full-length cDNA encoding a novel sodium channel and 25.1 extended from domain H to the 3-UTR. However, each had a deletion which truncated the coding region. Clone 1.18 had the 3‘- untranslated region, in addition to the C-terminus of the deduced amino acid sequence of PN5. The construct in the expression vector, pBSTACHr, consisted of sequences from 26.2 and 1.18.

PN5 homology to other known sodium channels was obtained using the GAP/Best Fit (GCG) program: Channel % Similarity % Identity PN3a 71 54 hPN3 71 55 PN4 71 53 PN4a 71 53 PN1 72 55 rat brain type I 72 55 rat brain type H 71 54 rat brain type ΙΠ 71 54 rat cardiac channel 73 56 rat skeletal muscle channel 71 53 Stabilizing the PN5 full length cDNA A. Media, E. coli cell lines, and growth conditions: Growth of fragments of PN5 could be accomplished under standard conditions; however growth of plasmids containing full length constructs of PN5 (in pCIneo, pBSTAcHr, and other vectors) could not be accomplished without use of special growth media, conditions, and E. coli strains. The following proved to be optimal: (1) use of E. coli STBL2™ for primary transformation following ligation reactions and for large scale culturing; (2) solid media was l/2x EM (see below) plus lx LB (Tryptone, 1%, Yeast Extract, 0.5%, NaCl, 0.5%), plus 15g/L agar, or lxFM plus l/2x LB; (3) liquid media optimally was lx FM plus l/2x LB; (4) carbenicillin, 100pg/ml, was used for all media, as it is metabolized less rapidly than ampicillin; (5) temperature for growth should be no greater than 30°C, usually 24-26°C; this necessitated longer growth periods than normally employed, from 24 to 72 hours. 2x Freezing Medium (2xFM): K2HP04 12.6g Na3Citrate 0.9g MgSO4.7H20 0.18g (NH4)2SO4 1.8g KH2PO4 3.6g Glycerol 88g H20 qs to DL 2x FM and die remaining media components are prepared separately, sterilized by autoclaving, cooled to at least 60°C, and added together to form the final medium. Carbenicillin is prepared 25 at 25mg/ml H20 and sterilized by filtration. 2x FM was first described for preparation of frozen stocks of bacterial cells (Practical Methods in Molecular Biology, Schleif, R.F. and Wensink, P.C., Springer-Verlag, New York (1981) pp. 201-202).

B. Expression Vectors In order to provide for increased stability of the full length cDNA, the oocyte expression vector pBSTAcHr was modified to reduce plasmid copy number when grown in E. coli and to reduce possible read-through transcription from vector sequences that might result in toxic cryptic expression of PN5 protein, Brosius J., Gene 27,151-160(1984). pBSTAcIIr was digested with PvuII. The 755 bp fragment containing the T7 promoter, β-globin 5‘UTR, the multiple cloning site, β-globin 3'UTR, and T3 promoter was ligated to the 3.6 kb fragment containing the replication origin, ampicillin resistance gene, rmBTj and rmBTjTj transcription terminators from pKK232-8, which had been fully digested with Smal and partially digested with PvuH and treated with shrimp intestinal phosphatase to prevent self ligation. The resulting plasmid in which the orientation of the pBSTA fragment is such that the T7 promoter is proximal to the rmBTj terminator was identified by restriction mapping and named pHQ8. As is the case with pBSTA, the direction of transcription of the ampicillin resistance gene and replication origin of pHQ8 is opposite to that of the gene expression cassette, and the presence of the rmB Tj terminator should reduce any remaining read-through from the vector into the T7 promoter driven expression cassette.

C. Assembly of full length cDNA for expression _ ________ __ Since pBK-CMV.26.2 had a 58 bp deletion (corresponding to bp 4346 to 4403 of SEQ ID NO: 1) and the sequnce of pBK-CMV.1.18 begins at bp 4180 of SEQ ID NO: 1, pBKCMV.1.18 could be used to ,.repair pBK-CMV.26.2. A strategy was developed to assemble a full length cDNA from clones pBK-CMV.26.2 and pBK-CMV.1.18 in three sections, truncating the 5* and 3‘ UTRs and introducing unique restriction sites at the 5 ‘ and 3‘ ends in the process. The 5‘ end was generated by PCR from 26.2, truncating the 5‘ UTR by incorporating a Sail site just upstream of the start codon. The central section was a restriction fragment from 26.2. The 3' end was prepared by overlap PCR from both 26.2 and 1.18 and incorporating an Xbal site just down stream of the stop codon. These sections were digested at unique restriction sites and assembled in pBSTAcIIr. Although this construct appeared to have a correct sequence, upon recloning as a Sail to Xbal fragment into pCIneo, two type of isolates were found, one with a deletion and one with an 8 bp insertion. Reexamination of the pBSTAcIIr clone showed the sequence was „mixed“ in this region, so that the clone must have rearranged. The 8 bp insertion was found as a repeat of one of the members of an 8 bp duplication in the native sequence, forming a triple 8 bp repeat in the rearranged isolate. Numerous cloning attempts inevitably gave rise to this rearrangement Overlap PCR was used to introduce silent mutations into one of the 8 bp repeats, and a fragment containing this region was included when the PN5 coding region was assembled into HQ8, the low-copy number version of pBSTAcIIr, to give plasmid HR-1. This sequence proved to be stable (see Figures 5A-E, SEQ ID NO: 5).

The 5‘ end fragment was prepared by PCR using pBK-CMV.26.2 DNA as template and primers 4999 (CTTGGTCGACTCTAGATCAGGGTGAAGATGGAGGAG·. Sail site underlined, PN5 homology in italics, corresponding to bp 58-77 of SEQ ID NO: 1, initiation codon in bold) and 4927 (GGGTTCAATGTGGTTTTATCT, corresponding to bp 1067 to 1047 of SEQ ED NO: 1), followed by gel purification, digestion with Sail and Kpnl (Kpnl site at pb 1003-1008, SEQ ID NO: 1), and gel purification.

The central 3.1 kb fragment was prepared by digestion of pBK-CMV.26.2 DNA with Kpnl and Aatll (AatH site at 4133-4138), followed by gel purification.

The 3‘ end fragment was prepared as follows: PCR using primers 4837 (TCTGGGAAGTTTGGAAG, corresponding to bp 3613 to 3629 of SEQ ED NO: 1) and 4931 (GACCACGAAGGCTATGTTGAGG, corresponding to bp 4239 to 4218 of SEQ ID NO: 1) on pBK-CMV.26.2 DNA as template gave a fragment of 0.6 kb. PCR using primers 4930 (CCTCAACATAGCCTTCGTGGTC, comesponding to bp 4218 to 4239 of SEQ ID NO: 1) and 4929 (GTCTTCTAGATGAGGGTTCA GTCATTGTG. Xbal site underlined, PN5 homology in italics, corresponding to pb 5386 to 5365 of SEQ ID NO: 1, stop codon in bold) on pBK-CMV.1.18 DNA as template gave a fragment of 1.2 kb, introducing a Xbal site 7 bp from the stop codon. Thus the 3' end of the 4837-4931 fragment exactly complements the 5‘ end of the 4930-4929 fragment. These two fragments were gel purified and a fraction of each combined as template in a PCR reaction using primers 4928 (CAAGCCTTTGTGTTCGAC, corresponding to bp 4084 to 4101 of SEQ ID NO: 1) and 4929, to give a fragment of 1.3 kb. This fragment was gel purifed, digested with Aatll and Xbal, and the 1.2 kb fragment gel purified.

The 3‘ end fragment was cloned into Aatll and Xbal digested pBSTAcIIr. One isloate was digested with Sail and Kpnl and ligated to the 5 ‘ end fragment. The resulting plasmid, after sequence verification, was digested with Kpnl and Aatll and ligated to the central 3.1 kb fragment, to form pBSTAcIIr.PN5(clone 21). pBSTAcIIr.PN5 (clone 21) was digested with Sail and Xbal to release the 5.3 kb PN5 fragment which was cloned into Sail and Xbal digested pCIneoII. Multiple isolates were found, of which GPII-1, which was completely sequenced, was typical and contained an 8 bp insert. This CAGAAGAA, after pb 3994 of .. SEQ ID NO: 1, converted the direct repeat of this sequence at this location into a triple direct repeat, causing a shift in the reading frame. In an attempt to repair this defect, pBSTAcIIr. PN5 (clone 21) was digested with Nhel (bp 2538-2543 SEQ ID NO: 1) and Xhol (bp 48284833, SEQ ID NO: 1) to give a 6.2 kb fragment and with Aatll and Xhol to give a 0.7 kb fragment which were ligated to the 1.6 kp fragment resulting from digestion of pBKCMV.26.2 with Aatll and Nhel. Although no isolates were found which were completely correct, one isolate, HA-4, had only a single base change, deletion of the C at bp 4827 (SEQ ID NO: 1) adjacent to the Xhol site.

In order to prevent the 8 bp insertion rearrangement from occurring, three silent mutations were introduced in the 5' repeat, and two additional mutations in a string ot Ts would also be introduced, as shown below (bp 3982 to 4014, SEQ ID NO: 1; mutation sites underlined, 8 bp repeats in native sequence in italics): native GAC ATT TTT ATG ACA GAA GAA CAG AAG AAA TAT Asp He Phe Met Thr Glu Glu Gin Lys Lys Tyr mutant GAC ATC TTC ATG ACT GAG GAQ CAG AAG AAA TAT As isolate HA-4 had the native direct repeat sequence (as opposed to e.g. pBSTAcIIr.PN5 (clone 21)) and the region near the Xhol site defect would not be involved, it was used as template DNA for the following PCR reactions. Primer P5-3716S (CCGAAGCCAATGTAACATTAGTAATTACTCGTG, corresponding to pb 3684 to 3716, SEQ ID NO: 1) was paired with primer P5-3969AS (GQTCQTCAGTCATQAAQATGTCTTGGCCACCTAAC, correspoind to bp 4003 to 3969, SEQ ID NO: 1, mutated bases are underlined) to give a 320 bp product. Primer P5-4017S (GGCCAAGACATCTTQATGACTGAQGAGCAGAAGAAATATTAC, corresponding to bp 3976 to 4017, SEQ ID NO: 1; mutated bases are underlined) was paired with primer PS4247 AS (CTCAAAGCAAAGACTTTGATGAGACACTCTATGG, corresoinding to bp 4280 to 4247, SEQ ID NO: 1) to give a 305 bp product. The 3‘ end of the 320 bp fragment thus has a 28 bp exact match to the 5 * end of the 305 bp fragment. The two bands were gel purified and a fraction of each combined in a new PCR reaction with primers P5-3716S and P54247AS to give a 597 bp product, which was T/A cloned into vector pCRU. Isolate HO-7 was found to have the desired sequence. A four-way ligation was performed to assemble the fulllength, modified PN5: the oocyte expression vector HQ-8 ws digested with Sail and Xbal to give a 4.4 kb vector fragment; GPII-1 was digested wtih Sail and Mlul to give a 3.8 kb fragment containing the 5‘ half of PN5; HO-7 was digested with Mlul (bp 3866 to 3871, SEQ ID NO: 1) and AatH to give a 0.3 kb fragment containing the mutant 8 bp repeat region of PN5; GPII-1 was digested with AatH and Xbal to give the remaining 1.3 kb 3 ‘ portion of PN5. A portion of the ligation reaction was transformed into E. coli Stable 2 cells. Of the 9.6 kb isolates containing all four fragments, HR-1 was sequenced and found to have the desired 5.4 kb sequence. These isolates grew well and showed no tendency to rearrange. The sequence of this engineered version of PN5 is shown in Figures 5A-E (SEQ ID NO: 5).

EXAMPLE 8 HUMAN PN5 An 856 bp clone (Figure 3A, SEQ ID No.: 3) has been isolated from a human dorsal root ganglia (DRG) cDNA library that is most closely related to rat PN5 with 79% identity for the amino acid sequence. The human PN5 sequence spans the region between HIS1 and interdomain IH/IV which includes the fast inactivation gate (i.e., IFM) that is located within interdomain ΠΙ/IV, The human DRG cDNA library was constructed from lumbar 4 and 5 DRG total RNA that was randomly primed. First strand cDNA was synthesized with Superscript Π reverse transcriptase (GIBCO BRL) and the second strand synthesis with T4 DNA polymerase. EcoRI adaptors were ligated to the ends of the double stranded cDNAs and the fragments cloned into the ZAP Π vector (Stratagene). The library was screened with digoxigenin-labeled rat PN3, rat PNl and human heart hHl probes. Positive clones were sequenced and compared to known human and rat sodium channel sequences. Only the aforementioned clone was identified as human PN5 sequence. Channel % Similarity % Identity Human Brain (HBA) 76 69 Human Heart (hHl) 81 74 30 Human Atypical Heart 60 52 Human Skeletal Muscle 80 71 Human Neuroendocrine 78 71 Human PN3 77 70 Rat PN1 79 72 Rat PN3 78 71 Rat PN4 78 70 Rat PN5 86 79 Figure 3B compares the amino acid sequence of the hPN5 fragment with the rat PN5 amino acid sequence in the appropriate region.

EXAMPLE 9 TISSUE DISTRIBUTION BY RT-PCR Brain, spina] cord, DRG, nodose ganglia, superior cervical ganglia, sciatic nerve, heart and skeletal muscle tissue were isolated from anesthetized, normal adult male Sprague-Dawley rats and were stored at -80°C. RNA was isolated from each tissue using RNAzol (Tel-Test, Inc.). Random-primed cDNA was reverse transcribed from 500ng of RNA from each tissue. The forward primer (CAGATTGTGTTCTCAGTACATTCC) and the reverse primer (CCAGGTGTCTAACGAATAAATAGG) were designed from the 3‘-untranslated region to yield a 252 base pair fragment. The cycle parameters were: 94°C/2 min. (denaturation), 94°C/30 sec., 65°C/30 sec. and 72°C/lmin. (35 cycles) and 72°C/4 min. The reaction products were analyzed on a 4% agarose gel. -- -.......-........

A positive control and a no-template control were also included. cDNA from each tissue was also PCR amplified using primers specific for glyceraldehyde-3-phosphate ' dehydrogenase to demonstrate template viability, as described by Tso et al., Nucleic Acid Res. 13, 2485-2502 (1985).

Tissue distribution profile of rPN5 by analysis of RNA from selected rat tissues by RTPCR was as follows: Tissue RT-PCR (35 cycles) Brain + Spinal cord + DRG +++ Nodose ganglia +++ Superior cervical ganglia + Sciatic nerve Heart Skeletal muscle FI 1-untreated + Fll-treated + PN5 was also detected after only 25 cycles (24 + 1) in the same five tissues as above in the same relative abundance.

EXAMPLE 10 ANTIBODIES A synthetic peptide (26 amino acids in interdomain Π and ΠΙ - residues 977 to 1002) was conjugated to KLH and antibody raised in rabbits. The antiserum was subsequently affinity purified.

PN5 constitutes a subfamily of novel sodium channel genes; these genes are different from . those detectable with other probes (e.g., PEAF8 and PN3 probes).

Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity and understanding, it will be obvious that certain changes and modifications may be practiced within the scope of the appended claims.

SEQUENCE LISTING (1) 1) GENERAL INFORMATION: (i) APPLICANT: (A) NAME: F. HOFFMANN-LA ROCHE AG (B) STREET: Grenzacherstrasse 124 (C) CITY: Basle (D) STATE: BS (E) COUNTRY: Switzerland (F) POSTAL CODE (ZIP): CH-4010 (G) TELEPHONE: 061-6884256 (H) TELEFAX: 061-6881395 (i) TELEX: 962292/965542 hlr ch (ii) TITLE OF INVENTION: Nucleic Acid Encoding a Nervous Tissue Sodium Channel (iii) NUMBER OF SEQUENCES: 5 (iv) COMPUTER READABLE FORM: (A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS (D) SOFTWARE: Patcntln Release # 1.0, Version # 1.30 (2) INFORMATION FOR SEQ ED NO: 1: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 5908 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: rat (F) TISSUE TYPE: Dorsal root ganglia (G) CELL TYPE: Peripheral nerve (xi) SEQUENCE DESCRIPTION: SEQ ED NO: 1: GAAGTCACAG GAGTGTCTGT CAGCGAGAGG AAGAAGGGAG AGTTTACTGA GTGTCTTCTG CCCCTCCTCA GGGTGAAGAT GGAGGAGAGG TACTACCCGG 33 TGATCTTCCC GCTGCCATAG AGACAAGGCG CCTCCAGGAA GCGAAGCCTC CATGGTGTTG CCTTGTTCAT ATCTCTGTCC CAACTGTATG TTCCCGAATA ATATTGGCAA GTGGAACTGG TTCCGGGCAG AGAGCTCTGA TGCCCTGCTG TCTTCTGCCT ATTCTGAACC CAACAAGGAT GTGGTACCTG AAAACCACAT CTGGTCCTTT GGCTTTACCG TTCGTGGTGG GGCTGTTGTC AGACAGAGGC GAGGAGAAGG TTCCCTTCAA GGACGAGCGG AGAAGCGGAT GCAGCTGAGC GTTACCTAAG TGGAAGACCT AACAAGAAGA TCTGGGGCCT ATTCAGTCTT TTCATGGCGA CGTCTTCATT GAGGCTTCAT CTGGACTTCA CCAAGTCAAT AGGCGATTTC CGCTCGGTGA CAGCATCTTT AGAAGTGTAT TGCTTTGAAA GCTCGGCAGC TGAACCCAGA CTCGCCATGT ACAGATCCTG TCATCTTCCT ACCATGGCTT CAAGGAGAAA AGGCTCTGGT GCTTCATCCT AATTTCCGCC TGCTATCCAA CCCAGCCTCG CTTTATGGTG GGACCCATTC GAACAATTTA TTTAATCCCC TAGCATGTTC ATTCTATGGA GGGATTTATA TGTGGATGAG TTGTCATTGG CTTTCAGCTC AGTTATCTCA AGAAGCTGGT GCCCTGGTCG TAAGCACAAC AGGAAAAAGA AGACCCTGTC CAATAATTAT TCCGGGTTAT CGGACCTCTG GGGCTCCTTC ATGAAGAACA ATGTTTCAGG TGCCATGGGA TTTCCCCGAA CCTTCACTTC AAGGAGAGGA GCCTCAGCTT ACATTCCCCC TACAAAGACC TCGCTTCAGC TCAGAAGCTT ATCATCTGCA GAGAAGTTTC TTTTAGAAGC TTTTCCTTCC AACAGCGATC TTCGTACCTT GGTCTGAAGG AGACGTGATG GTCAGCAGCT TGTGGCCCCA TAGCGAAGAC CCAATGGTTC ACAAAGTTTG GACTCAAGAC GGATCTACTT TACCTGCTTA GAACAGAAAT AAGCCCAGCA ATTGACAGAA GAAGAGGAAG CGACTCTCTG AGAAGTCCAA GACCTAAAGG TGAGCTTGTA ATAAGACATT GCCAAGCGGG AATGATTCGT CGGTGATCAT GACAACGACA TGTGATTAAA TCCGAGATCC GCAACTTGTT CCGAGTGTTC TCATCGTAGG GTCCTCACTC GTTCATGGGA ACCCTGCATC TTCATAATGT TACGTGCGAT ACAACTTTGG TCCTGGGAGA TGTCTTCTTC ACCTAACCCT GTAGCTGCTG GCTGTTAAGG GTTCCCTTAA TTTTTCGGTA 1451 GTAAGACAAG AAAGTCCTTC TTTATGAGAG GGTCCAAGAC GGCCCAAGCC 1501 TCAGCGTCTG ATTCAGAGGA CGATGCCTCT AAAAATCCAC AGCTCCTTGA 1551 GCAGACCAAA CGACTGTCCC AGAACTTGCC AGTGGATCTC TTTGATGAGC 1601 ACGTGGACCC CCTCCACAGG CAGAGAGCGC TGAGCGCTGT CAGTATCTTA 1651 ACCATCACCA TGCAGGAACA AGAAAAATTC CAGGAGCCTT GTTTCCCATG 1701 TGGGAAAAAT TTGGCCTCTA AGTACCTGGT GTGGGACTGT AGCCCTCAGT 1751 GGCTGTGCAT AAAGAAGGTC CTGCGGACCA TCATGACGGA TCCCTTTACT 1801 GAGCTGGCCA TCACCATCTG CATCATCATC AATACCGTTT TCTTAGCCGT 1851 GGAGCACCAC AACATGGATG ACAACTTAAA GACCATACTG AAAATAGGAA 1901 ACTGGGTTTT CACGGGAATT TTCATAGCGG AAATGTGTCT CAAGATCATC 1951 GCGCTCGACC CTTACCACTA CTTCCGGCAC GGCTGGAATG TTTTTGACAG 2001 CATCGTGGCC CTCCTGAGTC TCGCTGATGT GCTCTACAAC ACACTGTCTG 2051 ATAACAATAG gtctttcttg GCTTCCCTCA GAGTGCTGAG GGTCTTCAAG 2101 TTAGCCAAAT CCTGGCCCAC GTTAAACACT CTCATTAAGA TCATCGGCCA 2151 CTCCGTGGGC GCGCTTGGAA ACCTGACTGT GGTCCTGACT ATCGTGGTCT 2201 TCATCTTTTC TGTGGTGGGC ATGCGGCTCT TCGGCACCAA GTTTAACAAG 2251 ACCGCCTACG CCACCCAGGA GCGGCCCAGG CGGCGCTGGC ACATGGATAA 2301 TTTCTACCAC TCCTTCCTGG TGGTGTTCCG CATCCTCTGT GGGGAATGGA 2351 TCGAGAACAT GTGGGGCTGC ATGCAGGATA TGGACGGCTC CCCGTTGTGC 2401 ATCATTGTCT TTGTCCTGAT AATGGTGATC GGGAAGCTTG TGGTGCTTAA 2451 CCTCTTCATT GCCTTGCTGC TCAATTCCTT CAGCAATGAG GAGAAGGATG 2501 GGAGCCTGGA AGGAGAGACC AGGAAAACCA AAGTGCAGCT AGCCCTGGAT 2551 CGGTTCCGCC GGGCCTTCTC CTTCATGCTG CACGCTCTTC AGAGTTTTTG 2601 TTGCAAGAAA TGCAGGAGGA AAAACTCGCC AAAGCCAAAA GAGACAACAG 2651 AAAGCTTTGC TGGTGAGAAT AAAGACTCAA TCCTCCCGGA TGCGAGGCCC 2701 TGGAAGGAGT ATGATACAGA CATGGCTTTG TACACTGGAC AGGCCGGGGC 2751 TCCGCTGGCC CCACTCGCAG AGGTAGAGGA CGATGTGGAA TATTGTGGTG 2801 AAGGCGGTGC CCTACCCACC TCACAACATA 35 GTGCTGGAGT TCAGGCCGGT GACCTCCCTC TGAAATGGAA CTCGAAAGAA GACCTGAATG GCAGCCAGAT ACAAAACAGA AAAACCTGCT CTTTGTTATT TCCCCAGCCG TTCACATTTA ATTCCGGAGG TGGTGGTGTC CGGACTCTGC AATGAAGGTT ATGTCTTGCT GTAAATTTAT AAATATGTAT TTAGTAATTA AATGCCTATC AATCATGAAT TTGAGGCGAA GGCTCCTTCT CAATCAGCAG AACAGAAGAA CAAAAGCCCA CCTGGTCACA TAAATATGAT CAGAGACCAA GTATTTTCTG GTCTGACGCA ATATCTTTAG AGATGCTTTC CAAGAGAAAG ACCAAATCGT CTGCTGAGCA GCCCCAAGTT TTTTCCTCCT TATTTCACCA TGTGCTCAGT GGGCCCTGAG GTCGTCTACG GGTCTGCCTC TTTCTGGGAA TTGGATTTTA CTCGTGGAAG TCGCCCTGCT GCTGCTGTCG CCTCTACGCG TTACCCTGAA CAGAAAAAGT ATATTACAAT TCCCAAGGCC AGCCAGGTCT TATCATGATG GCAGCTCACT AAGAAGATCT GTGAGCATGC AAATTTACAG CCAAGGGCCT TCCCCCTGGG GAAGCACAGC GTGGAGCGCT GAGAAATTAC GGAAATGATC GTGCCTGGTG CTCATGAATC ACCTCTGCGG CCCTGATCAG ATTTTCTGGC GTTTGGAAGG CCGAAGTTCC GTCCCGCAGG GCAAGTGGCA ATTCCAGAGA TATCTCTACT CCTCTTTATC TAGGTGGCCA GCAATGAAAA CCTGAACAAA TTGACGTCAT GCTGAATCTG AGCCCGGATG GCATTTAAGC TCTCGGAATG AAAACAGTTT TAGTTGTCAC TCCTGTGGTG TGGTTTGAGA GATATTTGAA TAAGGTGTAC CTGAAGTGGG CTGGCTTGAT TACCAAGCTT GCGCTGTCCC CGCCATACCT TCGTATTTTG TGCATTAACG GAACCGAAGC TCAACTTTGA ACCTATAAGG GAAAGACGAG TTGTGGTTTT GGTGTTATTA AGACATTTTT AGTTAGGAAC TGTCAAGCCT CATTCTGGGT CCGACCAGCC ACCAAGGGGT ATACAGAGTC CAGCACAATT CCCCCAAAAA TTTCTATGCC GAACATTCGG gtttcataat GATGTCAATC CGATAATATT TGGCCTTTGG TTCCTCATTG GAAGTCCTTC AGTTTGAAGG GCCATTCTCA TATCTTGGGA GGACAGACAT CAATGTAACA CAACGTGGGG GCTGGCTGGA CAGCCGGACT TATCATCTTC TTGACAACTT ATGACAGAAG CAAGAAACCT TTGTGTTCGA CTTATTGTCT CAAAGATGTG AAGAAAACCT AGAGTGTCTC GCTGGAACTT CTGGTTTCCC CAGAGTCGTC CTGCCCGGGG TCTCTCTTCA CATCTTTGGG ACGACATCTT CAGATAACCA GGCAAAAGAA AGATAGCCGT GTGGTCAACA GGAGGAGAGC AGGTCTGGGA GCCCTCTCTG GCCGAATAAG.

ACCGCCTCCA GGGGACTCCA TATGGAGGCC CCAAGAGGAA CGGAAACACA TTCATCGCAC CCAAGGTCAA CTGCCTCACA TGAACACAGG GGCGTCCATT AGAAGGGACT TTGATATCCT ATCAAAGTCT ATTTGATTGT GCTTGGAGGA CGCTTGGCTC AATCAGGACC ACATCGGTCT ATGAGCTGGT CAACTTCGAG CTTCGGCTGG CACTGCAACT CGTCTACTTC TGTACATCGC GAGGACCCTC GAAGTTTGAC ACTTTGCGGA TTTCAGTTTC TTGCATGGAT GCGGCTTGGA AACCCTTTTA GGAGGAGGAG TGGAGAAGAT CAGGTGTTTT GGTTCACAAT GCTTAGCCTC CCGTTCGATC TTTAAATGAC GCAAAGGACA CAACATAGCC TTGCTTTGAG GTGGTCGTGG CAGTGACATT GGATTGGTCG CTCCTCTTTG GCTGCTCTTC TTTCCAAAGT ACCTTTACGG CTGGGATACC CCTCCTCCCA GTCAGTTACA TGTGATCCTC TGGGAGAGGA CCCGAGGCGT CGCCCTGCCG TAGTGATGGA gttctctttg TACCATGAAA AGAAGCTCTA CAAGGCGCCG GGTCAAACTG GCAATGGAGA GACTGAACCC CAGCCTCTGG TGTGTTTTTG TCTTGGAAAG CCGACCATAA TTCGTGGTCA GCAACACTAC TTCTTTCTAT TCTTTCCCGC AATCCTCAGG CTTTGATGAT CTGGTGATGT GAAGAAGGGC GCAGCATGCT CTCCTCAACC AGACAGCTGT TCATCATCTC GAGAACTTCA CGACTTTGAA CGCAGTTCAT GAGCCGTTGC CTTGCCCATG CTTTCACTAC ACCATGATGG CGAGCCCATA CCGTCATCCA AGGCTGAAGG CTTGTCCAGC TCATCTCCAC CGAGCAGGCG GCTGAACGAG ATTTCATGTA CGGAAGGCCT TCTTTACCAT TTCACCAATG CATTAGTACC CCACGCTCTT CTGGTCCGGG GTCTCTCCCC TCATTTACGC TCCGGGATCG GTGCCTCTTC CCATGCTGGA CAGCAGCCGC CTTCCTCATC ACACAGCCAC ATCTTCTATG CCAGTATTCG GTGTGGCCAA GTGATGGGCG CAGGGTCCTC AGGAGAAGTT GTCACCACCA GAGGGCCTAC ACAGGTCAAG TTGGATGTGG CCCTACCTCA GCAGACTCAC GTGACAGGTT GAGAGATGTT GGAGGACAGT 5601 CCAACTTACA TAAAGATGAG AAACAAGAAG GAAAGATCCC AGGAAAACTT 5651 CAGATTGTGT TCTCAGTACA TCCCCCAATG TGTCTGTTCG GTGTTTTGAG 5701 TATGTGACCT GCCACATGTA GCTCTTTTTT GCATGTACGT CAAAACCCTG 5751 CAGTAAGTTG ATAGCTTGCT ACGGGTGTTC CTACCAGCAT CACAGAATTG 5801 GGTGTATGAC TCAAACCTAA AAGCATGACT CTGACTTGTC AGTCAGCACC 5851 CCGACTTTCA GACGCTCCAA TCTCTGTCCC AGGTGTCTAA CGAATAAATA 5901 GGTAAAAG (3) INFORMATION FOR SEQ ID NO:2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1765 amino acids (B) TYPE: amino acid (C) STRANDEDNESS:_ (D) TOPOLOGY: not relevant (ii) MOLECULE TYPE: protein (iii) HYPOTHETICAL: YES (vi) ORIGINAL SOURCE: (A) ORGANISM: rat (F) Ή5SUE TYPE: dorsal root ganglia (G) CELL TYPE: peripheral nerve (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2: Met Glu Glu Arg Tyr Tyr Pro Val lie Phe Pro Asp Glu Arg Asn Phe ' Arg Pro Phe Thr Ser Asp Ser lie Gin Lys Glu Arg Lys Lys 35 Gin Pro Arg Pro Gin Leu Asp 50 55 Leu Tyr Gly Asp lie Pro Pro 65 70 Leu Asp Pro Phe Tyr Lys Asp Lys Arg Thr lie Tyr Arg Phe 15 Leu Ala Ala He Glu Lys Arg lie Ala 25 30 Ser Lys Asp Lys Ala Ala Ala Glu Pro 40 45 Leu Lys Ala Ser Arg Lys Leu Pro Lys 60 Glu Leu Val Ala Lys Pro Leu Glu Asp 75 80 His Lys Thr Phe Met Val Leu Asn Lys 90 95 Ser Ala Lys Arg Ala Leu Phe lie Leu 105 100 110 Gly Pro Phe Asn Pro Leu Arg Ser Leu Met 120 He Arg He 125 Ser Val His 115 Ser Val Phe Ser Met Phe He He Cys Thr Val He He Asn Cys Met 130 135 140 Phe Met Ala Asn Ser Met Glu Arg Ser Phe Asp Asn Asp He Pro Glu 145 150 155 160 Tyr Val Phe He Gly He Tyr He Leu Glu Ala Val He Lys He Leu 165 170 175 Ala Arg Gly Phe He Val Asp Glu Phe Ser Phe Leu Arg Asp Pro Trp 180 185 190 Asn Trp Leu Asp Phe He Val He Gly Thr Ala He Ala Thr Cys Phe 195 200 205 Pro Gly Ser Gin Val Asn Leu Ser Ala Leu Arg Thr Phe Arg Val Phe 210 215 220 Arg Ala Leu Lys Ala lie Ser Val lie Ser Gly Leu Lys Val lie Val· 225 230 235 240 Gly Ala Leu Leu Arg Ser Val Lys Lys Leu Val Asp Val Met Val Leu 245 250 255 Thr Leu Phe Cys Leu Ser He Phe Ala Leu Val Gly Gin Gin Leu Phe 260 265 270 Met Gly lie Leu Asn Gin Lys Cys He Lys His Asn Cys Gly Pro Asn 275 280 285 Pro Ala Ser Asn Lys Asp Cys Phe Glu Lys Glu Lys Asp Ser Glu Asp 290 295 300 Phe lie Met Cys Gly Thr Trp Leu Gly Ser Arg Pro Cys Pro Asn Gly 305 310 315 320 Ser Thr Cys Asp Lys Thr Thr Leu Asn Pro Asp Asn Asn Tyr Thr Lys 325 330 335 Phe Asp Asn Phe Gly Trp Ser Phe Leu Ala Met Phe Arg Val Met Thr 340 345 350 Gin Asp Ser Trp Glu Arg Leu Tyr Arg Gin He Leu Arg Thr Ser Gly 355 360 365 He Tyr Phe Val Phe Phe Phe Val Val Val He Phe Leu Gly Ser Phe 370 375 380 Tyr Leu Leu Asn Leu Thr Leu Ala Val Val Thr Met Ala Tyr Glu Glu 385 390 395 400 Gin Asn Arg Asn Val Ala Ala Glu Thr Glu Ala Lys Glu Lys Met Phe Gin Glu Ala Met Gly He 435 Ser Pro Lys 450 Phe Met Arg 465 Asp Asp Ala Ser Gin Asn His Arg Gin 515 Gin Glu Gin 530 Leu Ala Ser 545 lie Lys Lys Ala He Thr His His Asn 595 Trp Val Phe 610 Ala Leu Asp 625 Ser He Val Ser Asp Asn Phe Lys Leu 675 He Gly His 690 405 Gin Gin Leu 420 Asp Arg Ser Lys Arg Lys Gly Ser Lys 470 Ser Lys Asn 485 Leu Pro Val 500 Arg Ala Leu Glu Lys Phe Lys Tyr Leu 550 Val Leu Arg 565 He Cys lie 580 Met Asp Asp Thr Gly He Pro Tyr His 630 Ala Leu Leu 645 Asn Arg Ser 660 Ala Lys Ser Ser Val Gly 410 Leu Arg Glu Glu Lys 425 Ser Leu Asn Ser Leu 440 Phe Phe Gly Ser Lys 455 Thr Ala Gin Ala Ser 475 Pro Gin Leu Leu Glu 490 Asp Leu Phe Asp Glu 505 Ser Ala Val Ser He 520 Gin Glu Pro Cys Phe 535 Val Trp Asp Cys Ser 555 Thr He Met Thr Asp 570 He He Asn Thr Val 585 Asn Leu Lys Thr lie 600 Phe He Ala Glu Met 615 Tyr Phe Arg His Gly 635 Ser Leu Ala Asp Val 650 Phe Leu Ala Ser Leu 665 Trp Pro Thr Leu Asn 680 Ala Leu Gly Asn Leu 695 415 Glu Ala Leu Val Ala 430 Gin Ala Ser Ser Phe 445 Thr Arg Lys Ser Phe 460 Ala Ser Asp Ser Glu - 480 Gin Thr Lys Arg Leu 495 His Val Asp Pro Leu 510 Leu Thr He Thr Met 525 Pro Cys Gly Lys Asn 540 Pro Gin Trp Leu Cys 560 Pro Phe Thr Glu Leu 575 Phe Leu Ala Val Glu 590 Leu Lys He Gly Asn 605 Cys Leu Lys He He 620 Trp Asn Val Phe Asp 640 Leu Tyr Asn Thr Leu 655 Arg Val Leu Ajrg Val 670 Thr Leu He Lys He 685 Thr Val Val Leu Thr 700 He Val Val Phe He Phe Ser Val Val Gly Met Arg Leu Phe Gly Thr 705 710 715 720 Lys Phe Asn Lys Thr Ala Tyr Ala Thr Gin Glu Arg Pro Arg Arg Arg 725 730 735 Trp His Met Asp Asn Phe Tyr His Ser Phe Leu Val Val Phe Arg He 740 745 750 Leu Cys Gly Glu Trp He Glu Asn Met Trp Gly Cys Met Gin Asp Met 755 760 765 Asp Gly Ser Pro Leu Cys lie He Val Phe Val Leu lie Met Val He 770 775 780 Gly Lys Leu Val val Leu Asn Leu Phe He Ala Leu Leu Leu Asn Ser 785 790 795 800 Phe Ser Asn Glu Glu Lys Asp Gly Ser Leu Glu Gly Glu Thr Arg Lys 805 810 815 Thr Lys Val Gin Leu Ala Leu Asp Arg Phe Arg Arg Ala Phe Ser Phe 820 825 830 Met Leu His Ala Leu Gin Ser Phe Cys Cys Lys Lys Cys Arg Arg Lys 835 840 845 Asn Ser Pro Lys Pro Lys Glu Thr Thr Glu Ser Phe Ala Gly Glu Asn 850 855 860 Lys Asp Ser lie Leu Pro Asp Ala Arg Pro Trp Lys Glu Tyr Asp Thr 865 870 875 880 Asp Met Ala Leu Tyr Thr Gly Gin Ala Gly Ala Pro Leu Ala Pro Leu 885 890 895 Ala Glu Val Glu Asp Asp Val Glu Tyr Cys Gly Glu Gly Gly Ala Leu 900 905 910 Pro Thr Ser Gin His Ser Ala Gly Val Gin Ala Gly Asp Leu Pro Pro 915 920 925 Glu Thr Lys Gin Leu Thr Ser Pro Asp Asp Gin Gly Val Glu Met Glu 930 935 940 Val Phe Ser Glu Glu Asp Leu His Leu Ser He Gin Ser Pro Arg Lys 945 950 955 960 Lys Ser Asp Ala Val Ser Met Leu Ser Glu Cys Ser Thr lie Asp Leu 965 970 975 Asn Asp lie Phe Arg Asn Leu Gin Lys Thr Val Ser Pro Lys Lys Gin 980 985 990 Pro Asp Arg Cys Phe Pro Lys Gly Leu Ser Cys His Phe Leu Cys His 41 995 1000 1005 Lys Thr Asp Lys Arg Lys Ser Pro Trp Val Leu Trp Trp Asn He Arg 1010 1015 1020 Lys Thr Cys Tyr Gin He Val Lys His Ser Trp Phe Glu Ser Phe He 1025 , 1030 1035 1040 lie Phe Val lie Leu Leu Ser Ser Gly Ala Leu He Phe Glu Asp Val 1045 1050 1055 Asn Leu Pro Ser Arg Pro Gin Val Glu Lys Leu Leu Arg Cys Thr Asp 1060 1065 1070. Asn He Phe Thr Phe lie Phe Leu Leu Glu Met He Leu Lys Trp Val 1075 1080 1085 Ala Phe Gly Phe Arg Arg Tyr Phe Thr Ser Ala Trp Cys Trp Leu Asp 1090 1095 1100 Phe Leu lie Val Val Val Ser Val Leu Ser Leu Met Asn Leu Pro Ser 1105 i 1110 1115 1120 Leu Lys Ser Phe Arg Thr Leu Arg Ala Leu Arg Pro Leu Arg Ala Leu 1125 1130 1135 Ser Gin Phe Glu Gly Met Lys Val Val Val Tyr Ala Leu He Ser Ala 1140 1145 1150 lie Pro Ala He Leu Asn Val Leu Leu Val Cys Leu He Phe Trp Leu 1155 1160 1165 Val Phe Cys He Leu Gly Val Asn Leu Phe Ser Gly Lys Phe Gly Arg 1170 1175 1180 Cys He Asn Gly Thr Asp He Asn Met Tyr Leu Asp Phe Thr Glu Val 1185 ί 1190 1195 1200 Pro Asn Arg Ser Gin Cys Asn He Ser Asn Tyr Ser Trp Lys Val Pro 1205 1210 1215 Gin Val Asn Phe Asp Asn Val Gly Asn Ala Tyr Leu Ala Leu Leu Gin 1220 1225 1230 Val Ala Thr Tyr Lys Gly Trp Leu Glu He Met Asn Ala Ala Val Asp 1235 1240 1245 Ser Arg Glu Lys Asp Glu Gin Pro Asp Phe Glu Ala Asn Leu Tyr Ala 1250 1255 1260 Tyr Leu Tyr Phe Val Val Phe He He Phe Gly Ser Phe Phe Thr Leu 1265 ' 1270 1275 1280 Asn Leu Phe He Gly Val He He Asp Asn Phe Asn Gin Gin Gin Lys 1285 1290 1295 Lys Leu Gly Gly Gin Asp lie Phe Met Thr Glu Glu Gin Lys Lys Tyr 1300 1305 1310 Tyr Asn Ala Met Lys Lys Leu Gly Thr Lys Lys Pro Gin Lys Pro lie 1315 1320 1325 Pro Arg Pro Leu Asn Lys Cys Gin Ala Phe Val Phe Asp Leu Val Thr 1330 1335 1340 Ser Gin Val Phe Asp Val He He Leu Gly Leu He Val Leu Asn Met 1345 1350 1355 1360 He lie Met Met Ala Glu Ser Ala Asp Gin Pro Lys Asp Val Lys Lys 1365 1370 1375 . Thr Phe Asp He Leu Asn He Ala Phe Val Val He Phe Thr He Glu 1380 1385 1390 Cys Leu lie Lys Val Phe Ala Leu Arg Gin His Tyr Phe Thr Asn Gly 1395 1400 1405 Trp Asn Leu Phe Asp Cys Val Val Val Val Leu Ser He He Ser Thr 1410 1415 1420 Leu Val Ser Arg Leu Glu Asp Ser Asp He Ser Phe Pro Pro Thr Leu 1425 1430 1435 1440 Phe Arg Val Val Arg Leu Ala Arg He Gly Arg lie Leu Arg Leu Val 1445 1450 1455 Arg Ala Ala Arg Gly He Arg Thr Leu Leu Phe Ala Leu Met Met Ser 1460 1465 1470 Leu Pro Ser Leu Phe Asn He Gly Leu Leu Leu Phe Leu Val Met Phe 1475 1480 1485 He Tyr Ala He Phe Gly Met Ser Trp Phe Ser Lys Val Lys Lys Gly 1490 1495 1500 Ser Gly He Asp Asp He Phe Asn Phe Glu Thr Phe Thr Gly Ser Met 1505 1510 1515 1520 Leu Cys Leu Phe Gin lie Thr Thr Ser Ala Gly Trp Asp Thr Leu Leu 1525 1530 1535 Asn Pro Met Leu Glu Ala Lys Glu His Cys Asn Ser Ser Ser Gin Asp 1540 1545 1550 Ser Cys Gin Gin Pro Gin He Ala Val Val Tyr Phe Val Ser Tyr He 1555 - 1560 1565 He He Ser Phe Leu lie Val Val Asn Met Tyr lie Ala Val He Leu 1570 1575 1580 Glu Asn Phe Asn Thr Ala Thr Glu Glu Ser Glu Asp Pro Leu Gly Glu 1565 1590 1595 1600 Asp Asp Phe Glu lie Phe Tyr Glu Val Trp Glu Lys Phe Asp Pro Glu 1615 1605 1610 Ala Ser Gin Phe He Gin Tyr Ser Ala Leu Ser Asp Phe Ala Asp Ala 1620 1625 1630 Leu Pro Glu Pro Leu Arg Val Ala Lys Pro Asn Lys Phe Gin Phe Leu 1635 1640 1645 Val Met Asp Leu Pro Met Val Met Gly Asp Arg Leu His Cys Met Asp 1650 1655 1660 Val Leu Phe Ala Phe Thr Thr Arg Val Leu Gly Asp Ser Ser Gly Leu 1665 1670 1675 1680 Asp Thr Met Lys Thr Met Met Glu Glu Lys Phe Met Glu Ala Asn Pro 1665 1690 1695 Phe Lys Lys Leu Tyr Glu Pro He Val Thr Thr Thr Lys Arg Lys Glu 1700 1705 1710 Glu Glu Gin Gly Ala Ala Val He Gin Arg Ala Tyr Arg Lys His Met 1715 1720 1725 Glu Lys Met Val Lys Leu Arg Leu Lys Asp Arg Ser Ser Ser Ser His 1730 1735 1740 Gin Val Phe Cys Asn Gly Asp Leu Ser Ser Leu Asp Val Ala Lys Val 1745 1750 1755 1760 Lys Val His Asn Asp 1765 (4) INFORMATION FOR SEQ ID NO:3: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 856 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: Unear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: human (F) TISSUE TYPE: Dorsal root gangUa (G) CELL TYPE: Peripheral nerve (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: GCTGAGCAGT GGGGCACTGA TATTTGAAGA TGTTCACCTT GAGAACCAAC 44 CCAAAATCCA TTTATCCTGG TTTCACCAGT TGACCACCCT GCACTGAGGC GGTCAATGCT TCTGCCTCAT TCTGGAAAAT TACCATCATT TCAACCAGAA CTGCAAGTGG TGATTCCACA GTTACATTTA AATCTCTTCA GTTAGGTGGC ATGCAATGAA CCCGTT AGAATTACTA AGATGGTACT GCCTGGTGCT CATTAACTTA CTCTTCGTGC CTCATAGGTG TTTCTGGCTC TTGGGAAATG ACAAATAAAA AGTCAACTTT CAACATTTAA GAGAAAGAAC CTTCGTAGTC TTGGCGTTAT CAAGACATTT AAAATTAGGA AATTGTACTG AAAATGGGTA GCCTTGATTT ATGGAATTGA GCTGTCCCAG CCATACCTGC GTATTTTGTA CATTAATGGA GTCAATGTGA GACAATGTGG GGGCTGGATG AACAGCCAGA TTTATCATCT CATTGACAAC TTATGACAGA TCCAAAAAAC ACATTATTTT GCCTTCGGAT CATCATTGTG AGTCCTTCCG TTTGAAGGAA CATTCTGAAT TTCTGGGAGT ACAGACTCAG AAGTGGCAAT GAAATGCTTA GATATTATAT GTTTGAGAGC TTGGCTCATT TTCAACCAAC AGAACAGAAG CTCAAAAACC TACACATATT TTGGAAAGTA ATTGTCTCTG GACTCTACGA TGAAGGTGGT GTTTTGCTTG ATACTTCTTT TTATAAATTA TTCTCTTGGA CCTCGCTCTG ATGCAGCTGT AATTCACTCG CTTCACTCTG AGCAGAAAAA AAATACTATA CATTCCACGG (5) INFORMATION FOR SEQ ID NO:4: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 702 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: RT-PCR (A) DESCRIPTION: /desc = „DNA probe/domain IV“ (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: rat (F) TISSUE TYPE: dorsal root ganglia (G) CELL TYPE: peripheral nerve (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: CTCAACATGG GACGAAGGTT GCGAGTGTGT GGCTGGAACG GCTGTTTCT TCTTCCGGGT CGAGCAGCCA GCCCGCCCTC ACTCCATCTT ATCGACGACA GTTCCAGATC TCAACACGGG TCCCGGGGGA CTACATCATC TC TTACGATGAT CTGGGCAGAA GATGAAGATG TGTTCGACTT GCAATCCTTA CATCCGTCTG AGGGGATTCG TTCAACATCG CGGCATGGCC TGTTCAACTT ACCACCTCGG GCCTCCCTAC ACTGCGGGAG ATCTCCTTCC GGTGGAGACC TCAACCAGTT TTCGCCCTGC CATAGTGGTG AGTCACTGGA GCCAGGATCG CACGCTGCTC GCCTCCTCCT AGCTTCGCTA CAAGACCTTT CCGGCTGGGA TGCGACCCCA CCCGGCGGTG TCATCGTGGT GACGAGCAGG CTTTGTGGCC GACAGTACTA ATCCTGTCCA AAACTACTTC GCCGCATCCT TTCGCCCTCA CTTCCTCGTC ACGTCGTGGA GGCAACAGCA CGGCCTCCTC ACCTGCCCAA GGCATCATCT CAACATGTAT GCGAGGAGAA GTCTTCACGG TTTCACCAAC TTGGGAGTCT TCCCCGACGC CAGGCTGATC TGATGTCCCT ATGTTCATCT CGAGGC.CGGC TGCTGTGCCT AGCCCCATCC CAGCAACGGC TCTTCACCAC ATCGCAGTCA (5) INFORMATION FOR SEQ ID NO:5: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 5334 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: RT-PCR (A) DESCRIPTION: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: (F) TISSUE TYPE: (G) CELL TYPE: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: GTCGACTCTA TTCCCGGACG CATAGAGAAG AGGCGGCAGC AGGAAGTTAC GCCTCTGGAA TGTTGAACAA TTCATTCTGG TGTCCATTCA GTATGTTCAT GAATACGTCT GGCAAGAGGC ACTGGCTGGA GGCAGCCAAG TCTGAAGGCG TGCTGCGCTC TGCCTCAGCA GAACCAGAAG AGGATTGCTT ACCTGGCTCG GATCAGGGTG AGCGGAATTT CGGATTGCTA TGAGCCCCAG CTAAGCTTTA GACCTGGACC GAAGAGAACA GGCCTTTTAA GTCTTTAGCA GGCGAATTCT TCATTGGGAT TTCATTGTGG CTTCATTGTC TCAATCTTTC ATTTCAGTTA GGTGAAGAAG TCTTTGCCCT TGTATTAAGC TGAAAAGGAA GCAGCAGACC AAGATGGAGG CCGCCCCTTC TCCAAAAGGA CCTCGGCCTC TGGTGACATT CATTCTACAA ATTTATCGCT TCCCCTCAGA TGTTCATCAT ATGGAGAGAA TTATATTTTA ATGAGTTTTC ATTGGAACAG AGCTCTTCGT TCTCAGGTCT CTGGTAGACG GGTCGGTCAG ACAACTGTGG AAAGATAGCG CTGTCCCAAT AGAGGTACTA ACTTCCGACT GAGGAAGAAG AGCTTGACCT CCCCCTGAGC AGACCATAAG TCAGCGCCAA AGCTTAATGA CTGCACGGTG GTTTCGACAA GAAGCTGTGA CTTCCTCCGA CGATCGCAAC ACCTTCCGAG GAAGGTCATC TGATGGTCCT CAGCTGTTCA CCCCAACCCT AAGACTTCAT GGTTCTACGT CCCGGTGATC CTCTGGCTGC TCCAAAGACA AAAGGCCTCC TTGTAGCGAA ACATTCATGG GCGGGCCTTG TTCGTATCTC ATCATCAACT CGACATTCCC TTAAAATATT GATCCGTGGA TTGTTTTCCG TGTTCAGAGC GTAGGTGCCC CACTCTCTTC TGGGAATTCT GCATCCAACA AATGTGTGGT GCGATAAAAC CACATTGAAC CCAGACAATA ATTATACAAA GTTTGACAAC TTTGGCTGGT CCTTTCTCGC CATGTTCCGG GTTATGACTC AAGACTCCTG GGAGAGGCTT TACCGACAGA TCCTGCGGAC CTCTGGGATC- TACTTTGTCT TCTTCTTCGT GGTGGTCATC TTCCTGGGCT CCTTCTACCT GCTTAACCTA ACCCTGGCTG TTGTCACCAT GGCTTATGAA GAACAGAACA GAAATGTAGC TGCTGAGACA GAGGCCAAGG AGAAAATGTT TCAGGAAGCC CAGCAGCTGT TAAGGGAGGA GAAGGAGGCT CTGGTTGCCA TGGGAATTGA CAGAAGTTCC CTTAATTCCC TTCAAGCTTC ATCCTTTTCC CCGAAGAAGA GGAAGTTTTT CGGTAGTAAG ACAAGAAAGT CCTTCTTTAT GAGAGGGTCC AAGACGGCCC AAGCCTCAGC GTCTGATTCA GAGGACGATG CCTCTAAAAA TCCACAGCTC CTTGAGCAGA CCAAACGACT GTCCCAGAAC TTGCCAGTGG ATCTCTTTGA TGAGCACGTG GACCCCCTCC ACAGGCAGAG AGCGCTGAGC GCTGTCAGTA TCTTAACCAT CACCATGCAG GAACAAGAAA AATTCCAGGA GCCTTGTTTC CCATGTGGGA AAAATTTGGC CTCTAAGTAC CTGGTGTGGG ACTGTAGCCC TCAGTGGCTG TGCATAAAGA AGGTCCTGCG GACCATCATG ACGGATCCCT TTACTGAGCT GGCCATCACC ATCTGCATCA TCATCAATAC CGTTTTCTTA GCCGTGGAGC ACCACAACAT GGATGACAAC TTAAAGACCA TACTGAAAAT AGGAAACTGG GTTTTCACGG GAATTTTCAT AGCGGAAATG TGTCTCAAGA TCATCGCGCT CGACCCTTAC CACTACTTCC GGCACGGCTG GAATGTTTTT GACAGCATCG TGGCCCTCCT GAGTCTCGCT GATGTGCTCT ACAACACACT GTCTGATAAC AATAGGTCTT TCTTGGCTTC CCTCAGAGTG CTGAGGGTCT TCAAGTTAGC CAAATCCTGG CCCACGTTAA ACACTCTCAT TAAGATCATC GGCCACTCCG TGGGCGCGCT TGGAAACCTG ACTGTGGTCC TGACTATCGT GGTCTTCATC TTTTCTGTGG TGGGCATGCG GCTCTTCGGC ACCAAGTTTA ACAAGACCGC CTACGCCACC CAGGAGCGGC CCAGGCGGCG CTGGCACATG GATAATTTCT ACCACTCCTT CCTGGTGGTG TTCCGCATCC TCTGTGGGGA ATGGATCGAG AACATGTGGG GCTGCATGCA GGATATGGAC GGCTCCCCGT TGTGCATCAT TGTCTTTGTC CTGATAATGG TGATCGGGAA GCTTGTGGTG CTTAACCTCT 48 TCATTGCCTT CTGGAAGGAG CCGCCGGGCC AGAAATGCAG TTTGCTGGTG GGAGTATGAT TGGCCCCACT GGTGCCCTAC CCCTCCAGAG TGGAAGTATT AAGAAGTCTG GAATGATATC CAGATAGATG ACAGACAAGA CTGCTACCAA TTATTCTGCT AGCCGGCCCC ATTTATTTTC GGAGGTATTT GTGTCTGTGC TCTGCGGGCC AGGTTGTCGT TTGCTGGTCT TTTATTTTCT TGTATTTGGA AATTACTCGT CTATCTCGCC TGAATGCTGC GCTGCTCAAT AGACCAGGAA TTCTCCTTCA GAGGAAAAAC AGAATAAAGA ACAGACATGG CGCAGAGGTA CCACCTCACA ACCAAGCAGC TTCTGAAGAA ACGCAGTGAG TTTAGAAATT CTTTCCCAAG GAAAGTCCCC ATCGTGAAGC GAGCAGTGGA AAGTTGAGAA CTCCTGGAAA CACCAGTGCC TCAGTCTCAT CTGAGACCTC CTACGCCCTG GCCTCATTTT GGGAAGTTTG TTTTACCGAA GGAAGGTCCC CTGCTGCAAG TGTCGATTCC TCCTTCAGCA AACCAAAGTG TGCTGCACGC TCGCCAAAGC CTCAATCCTC CTTTGTACAC GAGGACGATG ACATAGTGCT TCACTAGCCC GATCTGCATT CATGCTCTCG TACAGAAAAC GGCCTTAGTT CTGGGTCCTG ACAGCTGGTT GCGCTGATAT ATTACTAAGG TGATCCTGAA TGGTGCTGGC GAATCTACCA TGCGGGCGCT ATCAGCGCCA CTGGCTCGTA GAAGGTGCAT GTTCCGAACC GCAGGTCAAC TGGCAACCTA AGAGAGAAAG ATGAGGAGAA CAGCTAGCCC TCTTCAGAGT CAAAAGAGAC CCGGATGCGA TGGACAGGCC TGGAATATTG GGAGTTCAGG GGATGACCAA TAAGCATACA GAATGCAGCA AGTTTCCCCC GTCACTTTCT TGGTGGAACA TGAGAGTTTC TTGAAGATGT TGTACCGATA GTGGGTGGCC TTGATTTCCT AGCTTGAAGT GTCCCAGTTT TACCTGCCAT TTTTGTATCT TAACGGGACA GAAGCCAATG TTTGACAACG TAAGGGCTGG ACGAGCAGCC GGATGGGAGC TGGATCGGTT TTTTGTTGCA AACAGAAAGC GGCCCTGGAA GGGGCTCCGC TGGTGAAGGC CCGGTGACCT GGGGTTGAAA GAGTCCTCGA CAATTGACCT AAAAAGCAGC ATGCCACAAA TTCGGAAAAC ATAATCTTTG CAATCTCCCC ATATTTTCAC TTTGGATTCC CATTGTGGTG CCTTCCGGAC GAAGGAATGA TCTCAATGTC TGGGAGTAAA GACATAAATA TAACATTAGT TGGGGAATGC CTGGAAATCA GGACTTTGAG GCGAACCTCT CTTCTTTACC AGCAGCAGAA AAGAAATATT GCCCATCCCA TCACAAGCCA ATGATTATCA AACCTTTGAT GTCTCATCAA AACTTATTTG TTCCCGCTTG TCGTCCGCTT CGGGGAATCA CTTCAACATC TTGGGATGAG ATCTTCAACT AACCACTTCG AAGAACACTG GCCGTCGTCT CAACATGTAC AGAGCGAGGA TGGGAGAAGT CTCTGACTTT ATAAGTTTCA CTCCATTGCA CTCCAGCGGC AGGCCAACCC AGGAAGGAGG ACGCGTATCT CTGAACCTCT AAAGTTAGGT ACAATGCAAT AGGCCCCTGA GGTCTTTGAC TGATGGCTGA ATCCTCAACA AGTCTTTGCT ATTGTGTGGT GAGGACAGTG GGCTCGGATT GGACCCTCCT GGTCTGCTGC CTGGTTTTCC TCGAGACCTT GCTGGCTGGG CAACTCCTCC ACTTCGTCAG ATCGCTGTGA CCCTCTGGGA TTGACCCCGA GCGGACGCCC GTTTCTAGTG TGGATGTTCT TTGGATACCA TTTTAAGAAG AGGAGCAAGG CTACTTTGTG TTATCGGTGT GGCCAAGACA GAAAAAGTTA ACAAATGTCA GTCATCATTC ATCTGCCGAC TAGCCTTCGT TTGAGGCAAC CGTGGTTCTT ACATTTCTTT GGTCGAATCC CTTTGCTTTG TCTTCCTGGT AAAGTGAAGA TACGGGCAGC ATACCCTCCT TCCCAAGACA TTACATCATC TCCTCGAGAA GAGGACGACT GGCGTCGCAG TGCCGGAGCC ATGGACTTGC CTTTGCTTTC TGAAAACCAT CTCTACGAGC CGCCGCCGTC GTTTTTATCA TATTATTGAC T£TT£ATGAC GGAACCAAGA AGCCTTTGTG TGGGTCTTAT CAGCCCAAAG GGTCATCTTT ACTACTTCAC TCTATCATTA CCCGCCCACG TCAGGCTGGT ATGATGTCTC GATGTTCATT AGGGCTCCGG ATGCTGTGCC CAACCCCATG GCTGTCAGCA ATCTCCTTCC CTTCAACACA TTGAAATCTT TTCATCCAGT GTTGCGTGTG CCATGGTGAT ACTACCAGGG GATGGAGGAG CCATAGTCAC ATCCAGAGGG TCTTCGGCTC AACTTCAATC 2GA£GA£CAG AACCTCAAAA TTCGACCTGG TGTCTTAAAT ATGTGAAGAA ACCATAGAGT CAATGGCTGG GTACCCTGGT CTCTTCAGAG CCGGGCTGCC TCCCCTCTCT TACGCCATCT GATCGACGAC TCTTCCAGAT CTGGAGGCAA GCCGCAGATA TCATCGTGGT GCCACGGAGG CTATGAGGTC ATTCGGCCCT GCCAAGCCGA GGGCGACCGC TCCTCGGGGA AAGTTTATGG CACCACCAAG CCTACCGGAA • i 5201 5251 5301 ACACATGGAG AAGATGGTCA AACTGAGGCT GAAGGACAGG TCAAGTTCAT CGCACCAGGT GTTTTGCAAT GGAGACTTGT CCAGCTTGGA TGTGGCCAAG GTCAAGGTTC ACAATGACTG &ACCCTCATC TAGA

Claims (17)

What is claimed is:
1. I. ' An isolated DNA sequence comprising the nucleotide sequence set forth in SEQ ID NO: 1 or SEQ ID NO:3. 5
2. 2. The DNA of Claim 1 wherein said DNA sequence is encoding a sodium channel protein or fragment thereof.
3. 3. The DNA of Claim 2 wherein said sodium channel protein is the α-subunit or fragment thereof.
4. 4. The DNA of Claim 3 wherein said sodium channel protein is tetrodotoxin-resistant. 10 5. The DNA of Claim 3 or 4 wherein said sodium channel protein is found in mammals. 6. The DNA of Claim 3 or 4 wherein said sodium channel protein is found in rat. 7. The DNA of Claim 3 or 4 wherein said sodium channel protein is found in human.
5. 5. The DNA of Claim I wherein said DNA is cDNA.
6. 6. 9. The DNA of Claim 1 wherein said DNA is synthetic DNA. 15 10. Expression vectors comprising the DNA of Claim 8. II. Expression vectors comprising the synthetic DNA of Claim 9.
7. 7. 12. Host cells transformed with the expression vectors of Claim 10.
8. 8. 13. Host cells transformed with the expression vectors of Claim 11.
9. 9. 14. A recombinant polynucleotide comprising a nucleic acid sequence derived from the DNA 20 sequence of Claim 1.
10. 10. 15. A sodium channel protein encoded by a DNA of Claims 1 to 9 or allelic variants thereof.
11. 11. 16. A tetrodotoxin-resistant sodium channel protein encoded by a DNA of Claims 1 to 9 or allelic variants thereof.
12. 12. 17. The protein of Claim 16 having the amino acid sequence set forth in SEQ ID NO:2. 25
13. 13. 18. A method for identifying inhibitors of tetrodotoxin-resistant sodium channel protein comprising contacting a compound suspected of being said inhibitor with sodium channel protein of claim 16 and measuring the activity of said expressed sodium channel protein.
14. 14. 19. Poly- 30
15. 15. 20. A diagnostic kit comprising a polynucleotide of claim 14 capable of specifically hybridizing to a tetrodotoxin-resistant sodium channel protein or fragment thereof.
16. 16. 21. The use of an isolated DNA sequence of Claims 1 to 9 for identifying a compound suspected of being an inhibitor of tetrodotoxin-resistant sodium channel protein.
17. 17. 22. The invention substantially as hereinbefore described especially with reference to the 35 foregoing Examples.