GB2332906A - Nucleic acids encoding TTX-resistant Na channel proteins - Google Patents

Abstract

Nucleic acid sequences which encode TTX-resistant Na channel proteins derived from rat and human dorsal root ganglia are described. The products are designated PN5. The production an antiserum against a synthetic peptide from PN5 is described.

Description

1 2332906 This invention relates generally to sodium channel proteins and

more particularly to a novel nucleic acid sequence encoding for a mammalian a-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 1:0 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 voltagesensitive sodium channel is one of the most studied. It is a trarismembrane protein that is essential 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 or( gan 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., ENIBOJ 14, 1084-1090 (1995) C, 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 H, Noda et al., Nature 320, 188- 192 (1986), Ila, Auld et al., Neuron 1, 449-461 (1988), and M, Kayano et al., FEBS Lett. 228, 187-194 (1988), rat 11.9.98/Ar/vh skeletal muscle (SkMl), Trimmer et al., Neuron 3, 33-49 (1989), rat NaCh6, Schaller et al., J. Neurosci. 15, 3231-3242 (1995), rat peripheral nerve sodium channel type 3 (rPN3), Sangameswaran et al., I Biol Chem. 271, 5953-5956 (1996), also called SNS, Akoplan er al., Nature 379, 257-262 (1996), rat atypical channel, Felipe et al., J. Biol. Chem. 269, 3012530131 (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 intemal repeats, or homologous domains (domains I- IV), having similar amino acid sequences. Each domain folds into six predicted and helical umsmembrane 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-,,ating. 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 othe-,, 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 20 discloses purified Na+ channels that have proven useful as therapeutic and diagnos c tools.

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

I.D -D 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 1? 1 1 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 TrX, which can be measured by the IC50 values: TEX-sensitive Na+ channels (IC50 = 1-30 nNI), TTX-insensitive Na+ channels (IC50 = 1-5 pM), and TTX- resistant Na+ channels (IC50! 50 pM).

TTX-insensitive action potentials were first studied in rat skeletal muscle (Redfern er al., Acta Physiol. Scand. 82, 70-78 (1971)). Subsequently, these action potentials were described in other mammalian tissues, including newborn mammalian skeletal muscle, 0 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 TTX resistant (IC50 - 100 gK sodium channel currents, as described in Roy et al., J. Neurosci. 12, 2104-2111 (1992). TI'X-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.

SUNY OF THE UVENTION The present invention provides novel purified and isolated nucleic acid sequences encoding mammalian, preferably TTX-resistant, nervous tissue sodium channel proteins that 1 3 1-11 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, hean or skeletal muscle. In presently preferTed forms, novel DNA sequences comprise cDNA sequences encoding rat nervous tissue sodium channel protein. One aspect of the present invention is the 5 a-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. SpecificaIly, two cDNA sequences together encode for the full length rat nervous tissue sodium channel.

ZP 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 TTXresistant sodium channel comprising administering an effective amount of a compound having an IC50 of 10 pM or less.

4 i 1 11" 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, desig ing therapeutics and gn 5 screening for therapies.

BRIEF DESCRIMON OF THE SEO 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 ID 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 (I IV); the putative transmembrane segments (SI-S6); the amino acid conferring resistance to TTX (); N-glycosylation sites (e); cAND-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 ED NO: 3).

Figure 3B depicts the arnino 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 ED 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.

r DETAELED DESCRIMON OF THE INVENMON The present invention relates to a purified and isolated nucleic acid sequence encoding 1 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 5 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 transcnptase in the presence of an oligonucleotide primer will furnish an RNA-DNA duplex which can be treated with RNase H, DNA polymerase, and DNA hgase 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 (x subunit 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 IA-E (SEQ ID NO: 1) designated herein as sodium channel type 5 (PN5). Figures IA-E depict the 5908 nucleotide cDNA construct comprising a ZP ID 5298-base (counting the stop codon) open reading frame (SEQ ID NO: 1). Nucleotide residue z:1 C, 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 SaIJ-XbaI clone C) 6 1 lacks most of the untranslated sequences, the 5298 nucleotide open reading frame beginning at 0 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 dudna growth in E. Coli.

0 The nucleotide sequence of SEQ ID NO: 1 (Figures IA-E) cqrresponds 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 rPN1, with 72% and rat brain types 1 and Ill, with 71.8% and 71.3% respectively. Homology to rPN3a, h.PN3, rPN4, rPN4a, rat brain type II and rat skeletal muscle are each approximately 70 to 71%.

Additionally, an 856 base pair clone (SEQ ID NO: 3) as shown in Figure 3A has been isolated from a human dorsal root ganglia (DRG),cDNA library" 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 MS1 and interdomain M/IV which includes the fast 15 inactivation gate (i.e., WM) that is located within interdomain rWV.

The term,cDNA library" refers to a collection of clones, usually in a bacteriophaP, 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 7TX-resistant sodium channel 20 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 ID NO: 2), exhibits the primary structural features of an a-subunit of a voltacre-aated, TTX-resistant n -- sodiumchannel. Shown in Figures 2G-H are the homologous domains (PIV); the putative transmembrane segments (SPS6); the amino acid conferring resistance to TTX (); N- C glycosylation sites (o); and cAlvT-dependent PKA phosphorylation sites (0). DNA sequences 7 r% 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 PN1 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 10,insensitive" 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, DIIS4 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 DIIIS4, 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 DIUS3, the transmernbrane 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 DIE is even shorter by a deletion' of 3 amino acids. This shorterung of the S3 and the linker loop has been confirmed by designing primers in the appropriate region of the sequence for an RT- C PCR experiment from rat DRG and sequencing the amplified DNA fragment. Such an CP 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-pnmed RTPCR) 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: I 8 1 (Figures 1A-E). PN5 mRNA was present in five of the tissues studied: brain, spinal cord, DRG, nodose ganglia, and superior cervical anglia. PN5 was not present in the remaining 9 gn 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.

4n 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 fra,(;Tnents 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 deoxyribonucleoti des. This term 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 affinit' chromatography. In addition, polypeptides can also be made by synthetic means which are well known in the art.

9 n 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 cc-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 F1 1, a fusion cell line designed from neonatal rat DRG fused with a mouse cell line, N 1 8TG, from Massachusetts General Hospital.

F1 1 responds to trophic agents, such as NGF, by extending dendrites. It was found that PN5 was present in both native F1 1 and F1 I treated with NGF, leading the applicants to believe that the sodium channel is natively expressed in F1 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.

i i 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 Bsubunits may ZD modulate the function of the channel. However, since the cc-subunit is all that is required for the channel to be fully functional, expression of the cDNA in SEQ ID NO: I (Figures 1A-E) will provide a fully functional protein. The gene encoding the BI -subunit in peripheral nerve tissue was found to be identical to that found in rat heart, brain and skeletal muscle. The cDNA of the BI-subunit is not described herein as it is well known in the art, see Isom et at., Neuron 12, 1183-1194 (1994). However, it is to be understood that by combining the known t sequence for the BI-subunit with the a-subunit sequence described herein, one may obtain complete PN5 voltage-gated, preferably TFX-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 C> C) 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.

n 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 transferTed DNA. Host cells can also be made to express protein by direct injection with exogenous cRNA 5 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 rephcation 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 TTXresistant sodium channel protein comprising contacting a compound suspected of being an inhibitor C, r z:1 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.

Z:1 12 1 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 0 50% decrease in PN5 activity, preferably greater than 70% decrease in PN5 activity, more 5 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 TrXresistant 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 'I-fX-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 15 human DRG cDNA library. Human DRG are obtained at autopsy. The frozen tissue is homogerized and the RNA extracted with guanidine isothiocyanate (Chirgwin er al. Biochemistry 18, 5294-5299, (1979)). The RNA is size-fractionated on a sucrose gradient to enrich for large mRNAs because the sodium channel (x-subunits are encoded by large (7-11 kb) transcripts. Double-stranded cDNA is prepared using the SuperScript Choice cDNA 20 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 II vector (Stratagene) followed by packaging into phage particles.

Phage are plated out on 150 mm plates on a lawn of XLI-Blue NW' bacteria 0 (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 bythe rat PN5 probes 13 1_---\ hybridizing to positive human clones at high stringency should be stronger than obtained with C 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 immunoglobuhn fraction.

Monoclonal antibodies are prepared by standard hybridoma cell technology based on that reported by Kohler and NEIstein in Nature 256, 495-497 (1975). Spleen cells are obtained from a host animal immunized with the PN5 protein or a frag ent thereof, optionally linked to I 'm 1 a cariier. Hybrid cells are formed by fusing these spleen cells with an appropnate myeloma cell line and cultured. The antibodies produced by the hybrid cells are screened for their ability to bind to expressed PN5 proteins.

14 i A number of screening techniques well known in the art, such as, for example, forward or reverse enzyme-linked irrimunosorbent 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 5 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 10 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 TTX- r) resistant, sodium channel (x-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 TIX-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.

5. 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 16 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- Schriiiedeberg's Archives of Pharmacology 347,125-132 (1993) and McNeal, E.T. et al., I 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 pM or less, preferably!I;1M. 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 JC50" 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 r 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.

17 (1-1 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 5 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 10No. 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. 19 pol yvinylpyrToli done per 500 ml) DRG: dorsal root gangha EDTA: Ethylenediaminetetraacetic acid, tetrasodiurn salt MEN: 20 mM MOPS, 1 mM EDTA, 5 mM sodium acetate, pH 7.0 MOPS: 3-(N-morphohno)propanesulfonic acid (Sig MI ,ma Che ical Company) PN5: peripheral ner-ve sodium channel 5 PNS: peripheral nervous system SDS: sodium dodecyl sulfate SSC: 150 mM NaCl, 15 mM sodium citrate, pH 7.0 18 1 SSPE: 80 mM NaCI, 10 mM sodium phosphate, 1 m.M ethylenediarninetetraacetate, pH 8.0 TEV: two electrode voltage clamp TrX: tetrodotoxin (Sigma Chemical Company) 19 1 EXAMEPLES The following Examples illustrate practice of the invention.

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

The oocyte expression vector plasn-d pBSTAcllr was constructed from pBSTA by insertion of a synthetic oligonucleotide linker; plasmid pKK232- 8 was obtained from Pharmacia Biotech (Piscataway, NJ); plasn-d pCRII was obtained from Invitrogen, San Diego, CA. Competent E. coli cell lines STBL2Tm and SURE@ were obtained from Gibco/BRL and Stratagene, respectively.

EXAMPLE I

OBTAD\TING RNA FROM RAT DRG. BRAIN AND SPINAL CORD Lumbar DRG No. 4 and No. 5 (LA 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. 156 159 (1987)). Total RNA (5 Lg of each sample) was dissolved in MEN buffer containing 50% C.7 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 ael 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 gglml) followed by photography under UV light.

C 1 (1 1 \_ i After electrophoresis, the gel was rinsed in USSC and the RNA was transferred to a Duralose membrane (Stratagene) with 20xSSC by capillary action; the membrane was baked 0 under vacuum at 8TC for 1 hour.

EXAlvIPLE 2 PROBE FROM R-AT BRAIN IIA A 32p-labeled cRNA probe complementary to nucleotides 4637-5868 of the rat brain 11A sodium channel (x-subunit sequence was synthesized in vitro with 77 RNA polymerase 10(Pharmacia) using pEAF8 template DNA, (Noda et al., Nature 320,188-192 (1986)) that had been linearized with BstEIIProtocols for each procedure mentioned above can be found in Molecular Cloning: A 1 1 C Laboratory Manual by Sambrook et al. (Cold Spring Harbor Laboratory Press, 2nd edition, 1989).

EXAMPLE3 HYBRIDIZA17ON OF RNA WITH THE PROBE FROM RAT BRAIN 11A The membrane of Example 1 was prehybridized 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 (1 -mg/ml) for 16 hours at 42C. 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/n-) with the 32 P-labeled cRNA probe (ca. 1-3xlO 6 cpm/m.1) 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 550C 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 -80T with intensifying screens for up to 2 weeks. 21 (7 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.

EXANTLE 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 ZD between known sodium channels in domain IV. The domain IV 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 351 401 451 501 551 601 651 701 novel domain IV sequence was as follows (SEQ DD NO: 4):

1 CTCAACATGG TTACGATGAT GGTGGAGACC GACGAGCAGG 51 GACGAAGGTT CTGGGCAGAA TCAAI-"CAGTT CTTTGTGGCC 101 GCGAGTGTGT GATGAAGATG TTCGCCCTGC GACAGTACTA 151 GGCTGGAACG TGTTCGACTT CATAGTGGTG ATCCTGTCCA 201 GCTGTTTCT GCAATCCTTA AGTCACTGGA AAACTACTTC 251 TCTTCCGGGT CATCCGTCTG GCCAGGATCG GCCGCATCCT 301 CGAGCAGCCA AGGGGATTCG CACGCTGCTC TTCGCCCTCA GCCCGCCCTC TTCAACATCG GCCTCCTCCT ACTCCATCTT CGGCATGGCC AGCTTCGCTA ATCGACGACA TGTTCAACTT CAAGACCTTT GTTCCAGATC ACCACCTCGG CCGGCTGGGA TCAACACGGG GCCTCCCTAC TGCGACCCCA TCCCGGGGGA ACTGCGGGAG CCCGGCGGTG CTACATCATC ATCTCCTTCC TCATCGTGGT TC This sequence was labeled with 32 P by random priming z:, 22 CTTCCTCGTC AWT=GGA WCAACAWA CGGCCTCCTC ACCTGWCAA WCATCATCT CAACATGTAT GWAGGAGAA GTCTTCACGG =CACCAAC TTGWAGTCT TCCCCGACGC CAGGWGATC TGATGTC= AT=CATCT WAGG=GC TGCTGTGCCT AG=CATCC CAGCAAWGC T=CACCAC ATWCAGTCA EXANTLE5 HYBRIDIZATION OF RNA WITH THE NOVEL SODWM CHANNEL Y-UTR PROBE A Northern blot was prepared with lOgg 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 32 P UTP. The blot was prehybridized for 2 hours at 65'C in a solution containing 5XSSC, 1X Denhardt's solution, 0.5% SDS, 50mM sodium phosphate, pH 7. 1, 10salmon sperm DNA (1mg/rril) 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 32P-labeled probe was added at 7.5xlO5 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 65T for 20 min. The blot was analyzed on a PhosphoImager (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.

EX6ARLE 6 CONSTRUCTION & SCREENING OF cDNA LIBRARY FROM RAT DRG An EcoRI-adapted cDNA hbrary was prepared from normal adult male SpragueDawley rat DRG poly(A)+ RNA using the SuperScript Choice System (GEBCO 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 4-- packaged with the Gigapack II XL lambda packaging extract (Stratagene). Similarly, a >2kb Z 0 DRG cDNA library was synthesized.

23 n Phage (3.5X,05) were screened by filter hybridization with a 32 P-labeled probe (rBlIa, bases 4637-5868 as follows of Auld er al., Neuron 1, 449- 461 (1988)). Filters were hybridized in 50% formaniide, 5X SSPE, 5X Denhardt's solution, 0.5% SDS, 250tg/rrJ sheared, denatured salmon sperm DNA, and 50 mM sodium phosphate at 42'C and washed in 0.5X 5 SSC/0.1% SDS at 50C.

Southern blots of EcoRI-digested plasmids were hybridized with the 32 Plabeled DNA probe, (SEQ ID NO: 4). The filters were then hybridized in 50% formarruide, 6X SSC, 5X Denhardt's solution, 0.5%, SDS, and 100Lg/ml sheared, denatured salmon sperm DNA at 42'C and were washed in 0.1X SSC/0. 1% SDS at 65"C.

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

EXANTLE 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 0 domain II to the Y-UTR. However, each had a deletion which truncated the coding region. Clone 1. 18 had the Y- untranslated region, in addition to the C-terminus of the deduced arnino acid sequence of PN5. The construct in the expression vector, pBSTACI1r, consisted of sequences from 26.2 and 1. 18.

PN5 homology to other known sodium channels was obtained using the GAP/Best Fit 25 (GCG) program:

Channel PN3a hPN3 PN4 PN4a % Similarity % Identity 71 71 71 71 54 55 53 53 24 0, PN1 72 55 rat brain type 1 72 55 rat brain type 11 71 54 rat brain type M 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, pBSTAc][1r, -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 STBL2TM for primary transformation following ligation reactions and for large scale culturing; (2) solid media was 1/2x FM (see below) plus 1x LB (Tryptone, 1%, Yeast Extract, 0.5%, NaCl, 0.5%), plus 15g/L agar, or 1xFM plus 1/2x LB; (3) liquid media optimally was lx FM plus 1/2x LB; (4) carbenicillin, 100gg/ml, was used for all media, as it is metabolized less rapidly than ampicillin; (5) temperature for growth should be no greater than 300C, usually 24-26'C; this necessitated longer growth periods than normally employed, from 24 to 72 hours.

2x Freezin Medium (2xFW:

K2HP04 Na3Citrate MgS04.7H20 (NH4)2S04 KH2P04 Glycerol H20 12.6g 0.9g 0.18g 1.8g 3.6g 88g qs to EL 2x FM and the remaining media components are prepared separately, sterilized by autoclaving to 0' cooled to at least 60T, and added together to form the final medium. Carbenicillin is prepared 13 at 25mg/mI H20 and sterilized by filtration. 2x FM was first described for preparation of frozen stocks of bacterial cells (Practical Methods in Molecular Biology, Schielf, 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 plasriiid 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, Broslus L, Gene 27, 151-160(1984). pBSTAcIlr 10was digested with PvuIL The 755 bp fragment containing the T7 promoter, B-globin 5'UTR, the multiple cloning site, B-globin 3'UTR, and T3 promoter was ligated to the 3.6 kb fragment contailUng the replication origin, ampicillin resistance gene, rriBT, and rnBTIT Z:1 2 transcription terminators from pKK232-8, which had been fully digested with SmaI and partially digested with PvuE 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 77 promoter is proximal to the mBT, 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 0 cassette, and the presence of the rmB TI 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, pB KCMV-1.18 could be used to,,repair" p13K-CMY.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 introducincy unique restriction sites at the 5' and 3' ends in 1 0 the process. The 5' end 26 1 0 was generated by PCR from 26.2, truncating the YUTIR by incorporating a SalI site just upstream of the start codon. The central section was a restriction fragment from 26.2. The 3' 0 end was prepared by overlap PCR from both 26.2 and 1. 18 and incorporating an XbaI site just 0 1 down stream of the stop codon. These sections were digested at unique restriction sites and assembled in pBSTMIr. Although this construct appeared to have a correct sequence, upon recloning as a SalI to XbaI fragment into pCIneo, two type of isolates were found, one with a deletion and one with an 8 bp insertion. Reexamination of the pBSTAcIlr 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 pBSTAcHr, to give plasmid BRA. 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; Sall site underlined, PN5 homology in italics, corresponding to bp 58-77 of SEQ ID NO: 1, initiation codon in bold) and 4927 (CTGGTTCAATGTGG=ATCT, corresponding to bp 1067 to 1047 of SEQ ID NO: 1), foIlowed by gel purification, digestion with SaU and KpnI (KpnI site at pb 1003-1008, SEQ ID NO: 1), and gel purification.

The central 3.1 kb fragment was prepared by digestion of pBK-CM^V.26.2 DNA with Kpnl and AaX (AatE site at 4133-4138), followed by gel purification.

The Yend fragment was prepared as follows: PCR using primers 4837 (TCTGGGAAGTITGGAAG, corresponding to bp 3613 to 3629 of SEQ ID NO: 1) and 4931 C 27 1 1 1-1 (GACCACGAAGGCTATGTTGAGG, corresponding to bp 4239 to 4218 of SEQ ID NO: 1) on p13K-CMV.26.2 DNA as template gave a fragment of 0.6 kb. PCR using primers 4930 0 (CCTCAACATAGCCTTCGTGGTC, corresponding to bp 4218 to 4239 of SEQ DD NO: 1) and 4929 (GTM:T_AGATGAGGG77CAGTCATTGTG, XbaI site underlined, PN5 homology in italics, corresponding to pb 5386 to 5365 of SEQ ID NO: 1, stop codon in bold) on p13K-CMY1.18 DNA as template gave a fragment of 1. 2 kb, introducing a XbaI site 7 bp =I 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 (CAAGCCMGTGTTCGAC, corresponding to bp 4084 to 4101 of SEQ IlD NO: 1) and 4929, to give a fragment of 1.3 kb.

This fragment was gel purifed, digested with AatII and Xbal, and the 1.2 kb fragment gel purified.

The 3' end fragment was cloned into AatH and XbaI digested pBSTAcUr. One isloate was digested with SalI and KpnI and ligated to the 5' end fragment. The resulting plasmid, after sequence verification, was digested with KpnI and AatII and ligated to the central 3.1 kb fragment, to form pl3STAcUr.PN5 (clone 21). pBSTAcUr.PN5 (clone 21) was digested with SalI and XbaI to release the 5. 3 kb PN5 fragment which was cloned into SalI and XbaI digested pCIneol:L Multiple isolates were found, of which GPIP1, which was completely sequenced, was typicaland 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, pl3STAclIr. PN5 (clone 2 1) was digested with NheI (bp 2538-2543 SEQ ID NO: 1) and XhoI (bp 48284833, SEQ ID NO: 1) to give a 6.2 kb fragment and with AatE and)ChoI to give a 0.7 kb 1:5 fragment which were ligated to the 1.6 kp fragment resulting from digestion of pBK- C g-:, CMV.26.2 with AaW and NheL Although no isolates were found which were completely 1 correct, one isolate, HA-4, had only a single base C_ 28 1 c change, deletion of the C at bp 4827 (SEQ ID NO: 1) adjacent to the XhoI 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 ED NO: 1; mutation sites underlined, 8 bp repeats in native sequence in italics):

native GAC ATT M ATG ACA GAA GAA CAG AAG AAA TAT Asp Ile Phe Met Thr Glu Glu G1n Lys ' Lys Tyr mutant GAC ATC TTC ATG ACT GAG GAG CAG AAG AAA TAT As isolate HA-4 had the native direct repeat sequence (as opposed to e.g.

pBSTAclIr.PN5 (clone 21)) and the region near the XlioI site defect would not be involved, it was used as template DNA for the following PCR reactions. Primer P5-3716S (CCGAAGCCAATGTAACATTAGTAATrACTCGTG, corresponding to pb 3684 to 3716, 1 SEQ ID NO: 1) was paired with primer PS-3969AS (G:TCCTCAGTCATGAAGATGTMGGCCACCTAAC, correspoind to bp 4003 to 3969, SEQ ID NO: 1, mutated bases are underlined) to give a 320 bp product. Primer P5-4017S (GGCCAAGACATCTTCATGACTGA.GGAGCAGAAGAAATATFAC, corresponding to bp 3976 to 4017, SEQ ED NO: 1; mutated bases are underlined) was paired with primer P5 4247AS (CTCAAAGCAAAGAC=GATGAGACACTCTATGG, 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 P5 4247AS to give a 597 bp product,which was T/A cloned into vector pCRIL Isolate HO-7 was found to have the desired sequence. A four-way Egation was performed to assemble the full 0 length, modified PN5:

29 1 1 1, 1 the oocyte expression vector HQ-8 ws digested with Sall and Xbal to give a 4.4 kb vector C, fragment; GPH-1 was digested wtih SalI and Mlul to give a 3.8 kb fragment containing the 5' t ID half of PN5; HO-7 was digested with MuI (bp 3866 to 3871, SEQ ED NO: 1) and AatU to give r) C' a 0.3 kb fragment containing the mutant 8 bp repeat region of PN5; GPE-1 was digested with AatE and XbaI to give the remaining 1.3 kb 3' portion of PN5. A portion of the ligation =1 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 4: 4n PN5 is shown in Figures 5A-E (SEQ ID NO: 5).

EXANTLE8 HU 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 ECIS1 and interdomain EMV which includes the fast inactivation gate (i.e., IFW that is located within interdomain M/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 E 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 II vector (Stratagene). The library was screened with digoxigenin- labeled rat PN3, =- 4:1 rat PN1 and human heart hH1 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 (BBA) 76 69 Human Heart (hHI) 81 74 I 1 Human Atypical Heart 60 52 Human Skeletal Muscle 80 71 Human Neuroendocrine 78 71 Human PN3 77 70 Rat PN 1 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 DISTRIBLMON BY RT-PCR Brain, spinal cord, DRG, nodose ganglia, superior cervical ganglia, sciatic nerve, heart and skeletal muscle tissue were isolated ftom anesthetized, normal adult male Sprague-Dawley rats and were stored at -800C. RNA was isolated from each tissue using RNAzol (Tel-Test, 4D Inc.). Random-primed cDNA was reverse transcribed from 50Ong of RNA from each tissue.

The forward primer (CAGATTGTGTrCTCAGTACATTCC) and the reverse primer (CCAGGTGTCTAACGAATAAATAGG) were designed from the Y-untranslated region to yield a 252 base pair fragment. The cycle parameters were: WC/2 min. (denaturation), WC/30 sec., 65"C/30 sec. and 72"Cllmin. (35 cycles) and 721C/4 min. The reaction products were analyzed on a 4% agarose gel.

A positive control and a no-template control were also included. cIDNA from each tissue was also PCR amplified using primers specific for glyceraldehyde-3- phosphate 0 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 RT- PCR was as follows..

Tissue Brain RT-PCR (35 cvcles) 31 Spinal cord DRG Nodose ganglia Superior cervical ganglia + Sciatic ner-ve Heari Skeletal muscle F 11 -untreated F1 1 -treated i i i i i i PN5 was also detected after only 25 cycles (24 + 1) in the same five tissues as above in the same relative abundance.

EXAIPLE 10 ANTEBODIES A synthetic peptide (26 amino acids in interdomain U and IH - residues 977 to 1002) was conjugated to KLH and antibody raised in rabbits. The antiserurn was subsequently affinity purified.

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

32 1 1 1 1 (1) 1) GENERAL INFORMATION:

SEQUENCE LISTING APPLICANT: (A) NAME: F. HOFFMANN-LA. ROCHE AG (B) STREET: Grenzacherstrasse 124 (c) CITY: Basle (D) STATE:BS (E) COUNTRY: Switzerland (F) POSTAL CODE (ZEP): CH-4010 (G) TELEPHONE: 061-6884256 (H) TELEFAX: 061-6881395 (1) TELEX: 9622921965542 hIr ch (ii) TITLE OF INVENTION: Nucleic Acid Encoding a Nervous Tissue Sodium

Channel (iii) NUMBER OF SEQUENCES: 5 (1v) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS (D) SOFTWARE: PatentIn Release # 1.0, Version # 1.30 (2) INFORMATION FOR SEQ ID 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 (xl) SEQUENCE DESCRIPTION: SEQ ID NO: 1:

1 GAAWCACAG GAGTGTCTGT CAGWAGAGG AAGAAGGGAG AWTTACTGA 51 GTGTCTTCTG CCCCTCCTCA WGTGAAGAT GGAGGAGAGG TACTAC=G 33 101 TGAT== GGAWAWGG AAT=CG= CWTCACTTC CGACTCTCTG 151 GCTGWATAG AGAAWGGAT TGWATCCAA AAGGAGAGGA AGAMTCCAA 201 AGACAMGW WAGWGAGC CCCAGC=G G=CAG= GACCTAAAW 251 CCTWAGGAA GTTACCTAAG CTTTATGGTG ACATTW= TGAWTTGTA 301 GCGAAGCCTC TWAAGACCT GGACCCATTC TACAAAGACC ATAAGACATT 351 CATGGTGTTG AACAAGAMA GAMAATTTA TW=CAGC WCAAWGGG 401 CCTTGTTCAT TCTGGGGCCT TTTAATCCCC TCAGAAWTT AATGATT= 451 ATC=G= ATTCAGT= TAGCATG= ATCATCMCA CWTGATCAT 501 CAAWGTAM TTCATGGCGA ATTCTATWA GAGAMT= GACAAWACA 551 TTCCCGAATA CGTCTTCATT WGATTTATA TTTTAGAAGC TGTGATTAAA 601 ATATTGWAA GAGG=CAT T=GATGAG TTTTCCTTCC TWGAGATCC 651 GTGGAMMG WGGACTTCA TTGTCATTGG AACAGWATC GCAAWT= 701 TTCC'1'7GGCAG CCAAWCAAT CTTTCAGCTC TTCGTACCTT WGAGT=C 751 AGAG=TGA AGGWATTTC AGTTATCTCA GGTCTUAGG TCATWTAGG 801 TGCCCTGCTG CWTCWTGA AGAAGCTGGT AGAWTGATG GT=CACTC 851 TC=MCCT CAWATC= GCCCTGGTCG GTCAGCAGCT GTTCATGGGA 901 ATT=AACC AGAAGTGTAT TAAWACAAC TGTGGCCCCA ACCCTWATC 951 CAACAAWAT T=TTGAAA AGGAAAAMA TAGWAAGAC TTCATAATGT 1001 GTGWACCTG G=GWAGC AGACWTWC CCAATGG= TAWTGWAT 1051 AAAACCACAT TGAAWCAGA CAATAATTAT ACAAAW=G ACAAWTTGG 1101 CTGGTCCTTT CTCGCCATGT TCCGGGTTAT GACTCAMAC TCCTGGGAGA 1151 GG=TACW ACAGATC= WGACC=G GGATWACTT TGTCTTCTTC 1201 TTCGTGGTGG TCATCTTCCT GGG==C TACCTGWTA ACCTAMCCT 1251 GGCTGTTGTC ACCATGG= ATGAMAACA GAACAGAAAT GTAWTGWG 1301 AGACAGAGGC CAAGGAGAAA ATG=CAGG AMCCCAWA GCTGTTAAGG 1351 GAGGAGAAW AGG=TWT TGWATGGGA ATTGACAGAA GTTCCCTTAA 1401 TTCCCTTCAA G=CATCCT TTTCCCCGAA GAAGAGGAAG TTTTTCGGTA 34 1 1' 1 1451 GTAAGACAAG AAAGTCCTTC TTTATGAGAG G=CAAGAC GGCCCAAWC 1501 TCAGWT= ATTCAGAGGA CGATGCCTCT AAAAATWAC AG==GA 1551 GCAGACCAAA WACTGT= AGAMTTG= AGTGGATCTC TTTGATGAGC 1601 ACGTGGACCC =CCACAGG CAGAGAWGC TGAGWC= CAGTATCTTA 1651 ACCATCACCA TWAGGAACA AGAAAAATTC CAGGAGCCTT GTTTCCCATG 1701 TGWAAAAAT TTGW=TA AGTACCTGW GTGGGACTGT AGCWTCAW 1751 GG=TWAT AAAGAAWTC CTGWGACCA TCATGAWGA TCCCTTTACT 1801 GAWTGWCA TCACCAT= CATCATCATC AATACCG= TCTTAGCCGT 1851 GGAWACCAC AACATWATG ACAMTTAAA GACCATACTG AAAATAGGAA 1901 ACTGWT= CACWGAATT TTCATAG= AAATGTG= CAAGATCATC 1951 GCG=GACC CTTACCACTA CTTCCGGCAC GG=GAATG =TTGACAG 2001 CATWTGWC CTCCTGAGTC TCWTGAMT G=TACAM ACACTGT= 2051 ATAMAATAG GTCTTTCTTG G==TCA GAGTGCTGAG GGTCTTCAAG 2101 TTAGWAAAT CCTGGCCCAC GTTAAACACT =CATTAAGA TCATCGG= 2151 CTCCGTGGGC G=TTGGAA ACCTGAC= GGTCCTGACT ATWTGG= 2201 TCATC=TC TGTGGTGGGC ATGWGC= TCWCACCAA GTTTAACAAG 2251 ACCGCCTAW CCACCCAGGA GCG=CAGG WGCG=GC ACATGGATAA 2301 TTTCTACCAC TCCTTCCTGG TGGTGTTCCG CATCCTCTGT GGGGAATGGA 2351 TWAGAMAT GTGW=GC ATWAGGATA TGGACGGCTC CCWT=GC 2401 ATCATTG= TTGTCCTGAT AATGWGATC GGGAAGCTTG TGWG=AA 2451 CCTCTTCATT GCCTTGCTGC TCAATTC= CAWAATGAG GAGAMGATG 2501 GGAG=GGA AGGAGAGACC AGGAAAAWA AAGTGCAGCT AGCCCTGGAT 2551 CGGTTCCGCC GGGCCTTCTC CTTCATGCTG CACWTC= AGAGT=M 2601 TTWAWAAA TWAGGAGGA AAAACT=C AAAWCAAAA GAGACAMAG 2651 AAAWTTTGC TGWGAGAAT AAWACTCAA T==GGA TGWAGGCW 2701 TGGAAGGAGT ATGATACAGA CATGW=G TACACTWAC AGGCWGGGC 2751 TCCGCTGGCC CCACTMCAG AGGTAGAGGA WATWGGAA TATTGTGWG 2801 AWGCWTGC CCTACCCACC TCACAACATA GTGCTGGAW TCAGGCCWT 1 2851 GACCTW= CAGAGACCAA GCAGCTCACT AG=GGATG ACCAAGGWT 2901 TGAAATWAA GTATTTT= AAGAAGATCT GCATTTAAW ATACAGAGTC 2951 =GAAAGAA GMTGAWCA GTGAWATGC TCTCGGAATG CAWACAATT 3001 GACCTGAAM ATA.C-T-TTAG AAATTTACAG AAAACAG= =CCAAAAA 3051 GCAGCCAGAT AGATG=M CCAAGGGCCT TAGT=CAC TTTCTATGCC 3101 ACAAAACAGA CAAGAGAAAG MCC=GGG TCCTGTGGTG GAACATTCGG 3151 AAAACCTGCT ACCAAAT= GAAWACAGC TGWTTGAGA GTTMATAAT 3201 CTTTGTTATT WGCTGAWA GTWAGCWT GATATTTGAA GAMTCAATC 3251 TCCCCAGCCG G=CAAWT GAGAAATTAC TAAWTGTAC CGATAATATT 3301 TTCACATTTA TTTTCCTCCT GGAAATGATC CTGAAGTGGG TGGCCTTTGG 3351 ATTCWGAGG TATTTCACCA GTGCCTGGM CTGWTTGAT TTCCTCATTG 3401 TGGTGGTGTC TWGCTCAW WCATGAATC TACCAAWTT GAAWC=C 3451 CGGACTCTGC GGGCCCTGAG ACCTCTGCGG GCGCTGTCCC AGTTTGAAGG 3501 AATGAAW= GTCGTCTACG CWTGATCAG CGCCATACCT WCATTWCA 3551 ATGTCTTGCT G=MCCTC ATT=TWC TCGTATTTTG TATCTTGWA 3601 GTAAATTTAT TTTCTGGGAA GTTTGGAAGG MCATTAMG GGACAGACAT 3651 AAATATGTAT TTGGATTTTA CCGAAGTTCC GAAWGA.AGC CAATGTAACA 3701 TTAGMATTA CT=GGAAG GTCCCGCAGG TCAACTTTGA CAAWTGGGG 3751 AATGWTATC TCGCCCTGCT GCAAWGWA ACCTAMAGG GCTGWMGA 3801 AATCATGAAT GCTGCTGTCG ATTWAGAGA GAAAGAWAG CAGWGGACT 3851 TTGAGGWAA CCTCTACGCG TATCWTACT TTGTGW= TATCATC= 3901 GGCTCCTTCT TTACWTGAA CCTCTTTATC GG=TATTA TTGACAA= 3951 CAATCAWAG CAGAAAAAW TAGGTGWCA AGACATT= ATGACAGAAG 4001 AMAGAAGAA ATATTACAAT GCAATGAAAA AGTTAGGAAC CAAGAAAWT 4051 CAAAAWCCA TCCCAAGGCC CCTGAMAAA TGTCAAGCCT TTGTGTTCGA 4101 CCTGWCACA AGCCAGG= TTGAWTCAT CATTCTGWT CTTATTGTCT 4151 TAAATATGAT TATCATGATG GCTGAATCTG WGACCAGCC CAAAGAT= 36 i 1 1 \_---1 4201 AAGAAAAWT TTGATATCCT CAMATAGCC TTCGTGGTCA =TTACCAT 4251 AGAGTGT= ATCAAAG= TTGCTTTGAG GCAACACTAC TMACCAATG 4301 GCTGGAACTT ATTMATTGT GTGGTCGTGG T==TAT CATTAGTACC 4351 CTGGTTTCCC G=GGAGGA CAGTGACATT TCTTTCCCGC CCACWT= 4401 CAGAGTC= CGCTTGGCTC GGATTGG= AATCCTCAGG CTGGTCCGGG 4451 CTGWWGGG AATCAGGACC CTCCTCTTTG =TGATGAT GTCTCTCCCC 4501 TCTCTCTTCA ACATCGG= GCTGCTCTTC CTGGTGATGT-TCATTTACGC 4551 CATC=GGG ATGAGCTGGT =CCAAAGT GAAGAAWGC TCCGGGATCG 4601 AWACATC= CAA=TWAG ACCTTTACW GCAGCATGCT GTGWTC= 4651 CAGATAMCA CTTCGGCTGG CTGWATACC CTCCTCAACC CCATG=GA 4701 WCAAAAGAA CACMCAAW CCTCCTCCCA AGACAGCTGT CAWAGWGC 4751 AGATAGCWT CGTCTACTTC WCAGTTACA TCATCAT= CTTCCTCATC 4801 GTGWCAMA TGTACATCGC TGTGATCCTC GAGAMTTCA ACACAGWAC 4851 GGAGGAGAGC GAGGACC= TGWAGAGGA WACTTTGAA ATC=TATG 4901 AGGTC-TWGA GAAGTTMAC CCCGAGGCGT WCAGTTCAT CCAGTAT= 4951 GCCCTCTCTG ACT=CGGA CGCCCTGCCG GAGCWTTGC GTGTGGCCAA 5001 G=AATAAG TTTCAGTTTC TAGTGATGGA CTTGWCATG GTGATGWW 5051 ACCGCCTCCA TTGCATGGAT GTTCTCTTTG =TCACTAC CAGGGTCCTC 5101 GGGGACTCCA GCGGCTTGGA TACCATGAAA ACCATGATGG AGGAGAAGTT 5151 TATWAGGCC AAW=TTA AGAAWTCTA WAGWCATA GTCACCACCA 5201 CCAAGAGGAA GGAGGAGGAG CAAGGCGCCG CWTCATWA GAGGGCCTAC 5251 CGGAAACACA TWAGAAGAT dGTCAAACTG AGGCTGAAGG ACAGGTCAAG 5301 TTCATWCAC CAGGMT= GCAATWAGA CTTGTCCAGC TTGGAMMG 5351 CCAAWTCAA G=CACAAT GACTGAA= TCATCTWAC CWTACCTCA 5401 WG=CACA GCTTAGCCTC CAG=CMG CGAGCAGGCG WAGACTCAC 5451 TGAACACAGG CCGTTCGATC TGTGTTTTTG GCTGAACGAG GTGACAGGTT 5501 GGCGTCCATT =AAATGAC T=GGAAAG ATTTCATGTA GAGAGATGTT 5551 AGAAWGACT GMAGGACA WGACCATAA CGGAAGGCCT GGAGGACAW 37 5601 CCAACTTACA TAAAGATGAG AAACAAGAAG GAAAGAT= AGGAAAA= 5651 CAGATTGMT =CAGTACA TCCCCCAATG TGTCTGTTCG GT=TTGAG 5701 TATWGACCT GCCACATGTA GCTCTTTTTT GCATGTACGT CAAAAC=G 5751 CAGTAAWM ATAWTTGCT ACGGGTGTTC CTACCAWAT CACAGAATTG 5801 WTGTATGAC TCAAACCTAA AAWATGACT CTGACTT= AGMAWACC 5851 WGACT=A GACk"3rCTCCAA TCTCTGTCCC AGGT=TAA WAATAAATA 5901 GWAAAAG (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) TISSUE TYPE: dorsal root ganglia (G) CELL TYPE: peripheral nerve (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:

Met Glu Glu Arg Tyr Tyr Pro Val Ile Phe Pro Asp Glu Arg Asn Phe 10 15 Arg Pro Phe Thr Ser Asp Ser Leu Ala Ala Ile Glu Lys Arg Ile Ala 25 30 Ile Gln Lys Glu A--g Lys Lys Ser Lys Asp Lys Ala Ala Ala Glu Pro 40 45 Gln Pro Arg Pro Gln Leu Asp Leu Lys Ala Ser Arg Lys Leu Pro Lys 55 60 Leu Tyr Gly Asp Ile Pro Pro Glu Leu Val. Ala Lys Pro Leu Glu Asp 70 75 so Leu Asp Pro Phe Tyr Lys Asp His Lys Thr Phe Met Val Leu Asn Lys 90 95 ys Arg Thr Ile Tyr Arg Phe Ser Ala Lys Arg Ala Leu Phe Ile Leu 110 38 1- 1, Gly Pro Phe Asn Pro Leu Arg Ser Leu Met Ile Arg Ile Ser Val His 120 125 Ser Val Phe Ser Met Phe Ile Ile Cys Thr Val Ile Ile Asn Cys Met 135 140 Phe Met Ala Asn Ser Met Glu Arg Ser Phe Asp Asn Asp Ile Pro Glu 150 155 160 Tyr Val Phe Ile Gly Ile Tyr Ile Leu Glu Ala Val Ile Lys Ile Leu 170 175 Ala Arg Gly Phe Ile Val Asp Glu Phe Ser Phe Leu Arg Asp Pro Trp 185 190 Asn Trp Leu Asp Phe Ile Val Ile Gly Thr Ala Ile Ala Thr cys Phe 200 205 Pro Gly Ser Gln Val Asn Leu Ser Ala Leu Arg Thr Phe Arg Val Phe 210 215 220 Arg Ala Leu Lys Ala Ile Ser Val Ile Ser Gly Leu Lys Val Ile 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 Ile Phe Ala Leu Val Gly Gln Gln Leu Phe 260 265 270 Met Gly Ile Leu Asn Gln Lys Cys Ile Lys His Asn Cys Gly Pro Asm 275 280 285 Pro Ala Ser Asn Lys Asp Cys Phe Glu Lys Glu Lys Asp Ser Glu Asp 290 295 300 Phe Ile 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 Gln Asp Ser Trp, Glu Arg Leu Tyr Arg Gln Ile Leu Arg Thr Ser Gly 355 360 365 -1y Ser Phe Ile Tyr Phe Val Phe Phe Phe Val Val Val Ile Phe Leu G 370 375 380 Tyr- Leu Leu Asn Leu Thr Leu Ala Val Val Thr Met Ala Tyr Glu G-Lu 385 390 395 400 Gln Asn Arg Asn Val Ala Ala Glu Thr Glu Ala Lys Glu Lys Met Phe 39 405 410 415 Gin Glu Ala Gin Gin Leu Leu Arg Glu Glu Lys Glu Ala Leu Val Ala 420 425 430 Met Gly Ile Asp Arg Ser Ser Leu Asn Ser Leu Gin Ala Ser Ser Phe 435 440 445 Ser Pro Lys Lys Arg Lys Phe Phe Gly Ser Lys Thr Arg Lys Ser Phe 450 455 460 Phe Met Arg Gly Ser Lys Thr Ala Gin Ala Ser Ala Ser Asp Ser Glu 465 470 475. 480 Asp Asp Ala Ser Lys Asn Pro Gin Leu Leu Glu Gin Thr Lys Arg Leu 485 490 495 Ser Gin Asn Leu Pro Val Asp Leu Phe Asp Glu His Val Asp Pro Leu 500 505 510 His Arg Gin Arg Ala Leu Ser Ala Val Ser Ile Leu Thr Ile Thr Met 515 520 525 Gin Glu Gin Glu Lys Phe Gin Glu Pro Cys Phe Pro Cys Gly Lys Asn 530 535 540 Leu Ala Ser Lys Tyr Leu Val Trp Asp Cys Ser Pro Gin Trp, Leu Cys 545 550 555 560 Ile Lys Lys Val Leu Arg Thr Ile Met Thr Asp Pro Phe Thr Glu Leu 565 570 575 Ala Ile Thr Ile Cys Ile Ile Ile Asn Thr Val Phe Leu Ala Val Glu 580 585 590 His His Asn Met Asp Asp Asn Leu Lys Thr Ile Leu Lys Ile Gly Asn 595 600 605 Trp Val Phe Thr Gly Ile Phe Ile Ala Glu Met Cys Leu Lys Ile Ile 610 615 620 Ala Leu Asp Pro Tyr His Tyr Phe Arg His Gly T--P Asn Val Phe Asp 625 630 635 640 Ser Ile Val Ala Leu Leu Ser Leu Ala Asp Val lieu Tyr Asn Thr Leu 645 650 655 Ser Asp Asn Asn Arg Ser Phe Leu Ala Ser Leu Arg Val Leu Arg Val 660 665 670 Phe Lys Leu Ala 1,YS Ser Trp Pro Thr Leu Asn Thr Leu Ile Lys Ile 675 680 685 lie Gly His Ser Val Gly Ala Leu Gly Asn Leu Thr Val Val Leu Thr 690 695 700 Ile Val Val Phe Ile 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 '7 3 0 735 Trp His Met Asp Asn Phe Tyr His Ser Phe Leu Val Val Phe Arg Ile 740 745 750 Leu Cys Gly Glu Trp Ile Glu Asn Met Trp Gly Cys Met Gin Asp Met 755 760 765 Asp Gly Ser Pro Leu Cys Ile Ile Val Phe Val Leu Ile Met Val Ile 770 775 780 Gly Lys Leu Val Val Leu Asn Leu Phe Ile 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 Ile Leu Pro Asp Ala Arg Pro Trp Lys Glu Tyr Asp Thr 865 870 875 880 Asp Met Ala Leu Tyr Thr Gly Gln 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 Ile Gin Ser Pro Arg Lys 945 950 955 960 Lys Ser Asp Ala Val Ser Met Leu Ser Glu Cys Ser Thr Ile Asp Le-u.

965 970 975 Asn Asp Ile 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 Ile Arg 1010 1015 1020 Lys Thr Cys, Tyr Gin Ile Val Lys His Ser Trp Phe Glu Ser Phe Ile 1025 1030 1035 1040 Ile Phe Val Ile Leu Leu Ser Ser Gly Ala Leu Ile 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 io7o Asn Ile Phe Thr Phe Ile Phe Leu Leu Glu Met Ile 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 Ile Val Val Val Ser Val Leu Ser Leu Met Asn Leu Pro Ser 1105 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 Ile Ser Ala 1140 1145 1150 Ile Pro Ala Ile Leu Asn Val Leu Leu Val Cys Leu Ile Phe Trp Leu 1155 1160 1165 Val Phe Cys Ile Leu Gly Val Asn Leu Phe Ser Gly Lys Phe Gly Arg 11-70 1175 1180 Cys Ile Asn Gly Thr Asp Ile Asn Met Tyr Leu Asp Phe Thr Glu Val 1185 1190 1195 1200 Pro Asn Arg Ser Gin Cys Asn Ile Ser Asn Tyr Ser '11'rp 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 Ile Met Asn Ala Ala Val Asp 1235 1240 1245 Ser Arg Glu Lys Asp Glu Gin Pro Asp Phe Glu Ala Asn Leu '-T'yr Ala 1250 1255 1260 Tyr Leu Tyr Plie Val Val Phe Ile Ile Phe Gly Ser Phe Phe Th-- Leu 1265 1270 1275 1280 Asn Leu Phe Ile Gly Val Ile Tle Asp Asn Phe Asn Gin Gin Gin Lys 1285 1290 1295 42 1 1 Lys Leu Gly Gly Gln Asp Ile 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 Ile 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 Ile Ile Leu Gly Leu Ile Val Leu Asn Met 1345 1350 1355 1360 Ile Ile Met Met Ala Glu Ser Ala Asp Gin Pro Lys Asp Val Lys Lys 1365 1370 1375 Thr Phe Asp Ile Leu Asn Ile Ala Phe Val Val Ile Phe Thr Ile Glu 1380 1385 1390 Cys Leu Ile 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 Ile Ile Ser Thr 1410 1415 1420 Leu Val Ser Arg Leu Glu Asp Ser Asp Ile Ser Phe Pro Pro Thr Leu 1425 1430 1435 1440 Phe Arg Val Val Arg Leu Ala Arg Ile Gly Arg Ile Leu Arg Leu Val 1445 1450 1455 Arg Ala Ala Arg Gly Ile Arg Thr Leu Deu Phe Ala Leu Met Met Ser 1460 1465 1470 Leu Pro Ser Leu Phe Asn Ile Gly Leu Leu Leu Phe Leu Val Met Phe 1475 1480 1485 Ile Tyr Ala Ile Phe Gly Met Ser Trp Phe Ser Lys Val Lys Lys Gly 1490 1495 1500 Ser Gly Ile Asp Asp Ile Phe Asn Phe Glu Thr Phe Thr Gly Ser Met 1505 1510 1515 1520 Le%i Cys Leu Phe Gin Ile 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 Ile Ala Val Val Tyr Phe Val Ser Ty-- Ile 1555 1560 1565 Ile Ile Ser Phe Leu Ile Val Val Asn Met Tyr Ile Ala Val Ile Leu 1570 1575 1580 Glu Asn Phe Asn Thr Ala Thr Glu Glu Ser Glu Asp Pro Leu Gly Glu 43 1585 1590 1595 1600 Asp Asp Phe Glu Ile Phe Tyr Glu Val TrP Glu Lys Phe Asp Pro Glu 1605 1610 1615 Ala Ser Gln Phe Ile Gln 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 Gln Phe Leu 1635 1640 1645 Val Met Asp Leu Pro Met Val Met Gly Asp Arg Leu His Cys Met Asp 16 50 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 1685 1690 1695 Phe Lys Lys Leu Tyr Glu Pro Ile Val Thr Thr Thr Lys Arg Lys Glu 1700 1705 1710 Glu Glu Gln Gly Ala Ala Val Ile Gln 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 Gln Val Phe Cys Asn Gly Asp Leu Ser Ser Leu Asp Val Ala Lys Val 1745 1750 1755 Lys Val His Asn Asp 1765 (4) INFORMATION FOR SEQ ID NO:3:

(1) SEQ UENCE CHARACTERISTICS: (A) LENGTH: 856 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single C:

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (11i) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE:

1760 (A) ORGANISM: human (F) TISSUE TYPE: Dorsal rootCant.,ha (G) CELL TYPE: Peripheral nerve (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1 1 GCTGAWAGT GGGWACTGA TATTTGAAGA T=CACCTT GAGAACCAAC 44

51 CCAAAATWA AGAATTACTA AATTGTACTG ACATTATT= TACACATATT 101 TTTATCCTGG AGATWTACT AAAATGWTA G=TWGAT TTGGAAAGTA 151 TTTCACCAGT GCCTGGTGCT GCCTTGATTT CATCATT= ATTGTCTCTG 201 TGACCACCCT CATTAACTTA ATWAATTGA AGT==G GACTCTAWA 251 WACTGAGGC CTCTTCGTGC GCTGTCCCAG TTTGAAWAA TGAAGGTGGT 301 GGTCAATG= CTCATAGGTG CCATACCTGC CATT=GAAT GTTTTGCTTG 351 TCTGCCTCAT TTTCTGGCTC GTATTTTGTA TTCTGGGAGT ATACT=TT 401 =GGAAAAT TTGWAAATG CATTAAMGA ACAGACTCAG TTATAAATTA 451 TACCATCATT ACAAATAAAA GTCAATGTGA AAGTGWAAT TTCTCTTGGA 501 TCAAWAGAA AGTCAACT= GACAAT=G GAAATGWTA CCTCGCTCTG 551 CMCAAGTGG CAACATTTAA GGGCTGGAM GATATTATAT ATGCAGCTGT 601 TGATTWACA GAGAAAGAAC AACAGWAGA =TGAGAGC AATTCACTW 651 GTTACATTTA CTTCGTAGTC TTTATCATCT TTGWTCATT CTTCACTCTG 701 AATC==A TTGWGTTAT CATTGACAAC TTCAACCAAC AWAGAAAAA 751 GTTAGGMGC CAAGACATTT TTATGACAGA AGAMAGAAG AAATACTATA 801 AMCAATGAA AAAATTAGGA TCCAAAAAAC CTCAAAAACC CATTCCACGG 851 CCCGTT _ 1 1 1 =AACATGG 51 GAWAAWTT 101 WGAGT=T 151 GG=GAMG 201 GCTGTTTCT 251 TCTTCCGGGT 301 WAWAGWA 351 GCCCGCCCTC 401 ACTWATCTT 451 ATWAWACA 501 GTTCCAGATC 551 TCAACAWGG 601 TCCCGGGGGA CTACATCATC TC 651 701 INFORMATION FOR SEQ ID NOA: (1) SEQUENCE CHARACTERISTICS: (A) LENGTH: 702 base pairs (B) TYPE: nucleic acid (ii) (C) STRANDEDNESS: single (D) TOPOLOGY: linear MOLECULE TYPE: RT-PCR (A) DESCRIPTION: /desc =,,DNA probe/domain IV" (iii) HYPOTHETICAL: NO (1v) 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 NOA:

TTAWATGAT GGTGGAGACC CTGGGCAGAA TCAAWAGTT GATGAAGATG TTWC=GC TGTTCGACTT CATAGTG= GCAATCCTTA AGTCACTWA CATCWT= GWAGGATW AGGWATTW CACWTGWC TTCAACATCG GCCTCCTCCT CGWATGWC AGCTTCGCTA TGTTCAACTT CAAGACCT= ACCACCTCW CCGGCTGGGA G==TAC TGWACWCA ACTGWGGAG CCCGGCGGTG ATCTCCTTCC TCATCGTGGT INFORMATION FOR SEQ ID NO: 5:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 5334 base pairs (B) TYPE: nucleic acid (C) STRANDWNESS: sincyle 0 46 GACGAWAGG CTTTGTGGCC GACAGTACTA ATCCTGTCCA AAACTACTTC GCWCATCCT TTCGCCCTCA CTTCCTCGTC AWT=GGA GGCAACAWA CGGCCTCCTC ACCTGWCAA GWATCATCT CAACATWAT GWAGGAGAA GTCTTCACGG MCACCAAC TTGWAGTCT TCCCCGACGC CAGGCTGATC TGATGTC= AT=CATCT WAGG=GC TGWGTGWT AG=CATCC CAGCAAWGC T=CACCAC ATWCAGTCA i 1 01) (D) TOPOLOGY: linear MOLECULE TYPE: RT-PCR (A) DESCRIPTION: cDNA (iii) HYPOTlUEMCAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (A) ORGANISM:

(F) TISSUE TYPE: (G) CELL TYPE:

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5 1 GTCGACTCTA GATCAGG= AAGATWAGG

TTCCCGGACG 101 CATAGAGAAG 151 AGGCGWAGC 201 AGGAAWTAC 251 G==GAA 301 =TGAACAA 351 TTCATTCTGG 401 TGTCCATTCA 451 GTAT=CAT 501 GAATACG= 551 WCAAGAGGC 601 ACTGWTGGA 651 GWAGG7CCAAG 701 TWGAAGGCG 751 TGCTGCGCTC 801 MCCTCAWA 851 GAMCAGAAG 901 AGGATTG= 951 ACCTGG=G AGWGAATTT WGATTGCTA TGAG=CAG CTAAWTTTA GACCTWACC GAAGAGAACA GG==AA G=TTAWA GGWAATTCT TCATTGWAT TTCATTGMG CTTCATTGTC TCAATCT= ATTTCAWTA WMAAGAAG TCTTTGCCCT TGTATTAAW TGAAAAWAA GCAWAGACC CCGCCCCTTC TCCAAAAGGA CCTCGGCCTC TGWGACATT CATTWACAA ATTTATCWT TCCCCTCAGA T=CATCAT ATWAGAGAA TTATATTTTA ATGAGTT= ATTGGAACAG AGCTCTTCGT TCTCAGGTCT CTGWAGAW GGTCGGTCAG ACAAWGMG AAAGATAGW CTGTCCCAAT 47 AGAGGTACTA ACT=GACT GAGGAAGAAG AWTTGACCT CCCCCTGAGC AGACCATAAG TCAG=CAA AGWTAATGA CTGCACGGTG GTTTCGACAA GAAWTGTGA CTTCCTCCGA WATWCAAC ACCT=GAG GAAWTCATC TGATGW= CAGCTGTTCA CCCCAACCCT AAGAWTCAT GG=TAWT CCCGGTGATC CTCTGGCTGC TWAAAGACA AAAGGCCTCC TTGTAGWAA ACATTCAMG GCGGGCCTTG TTCGTATCTC ATCATCAACT WACATT= TTAAAATATT GATCWTGGA TTGTTTTCCG TGTTCAGAGC GTAGGTGWC CACT==C TGWAATTCT WATCCAACA AATWGTGW WGATAAAAC 1001 CACATTGAAC CCAGACAATA ATTATACAAA G=GACAAC TTTGWTGW 1051 CCTTTCTCGC CATGTTCCGG GTTATGACTC AAGACTC= GGAGAGG= 1101 TAWGACAGA TCCTGCGGAC CTCTGGGATC TACTTTGMT TCTTCTTCGT 1151 GGMGTCATC TTCCTGGGCT CCTTCTACCT G=AAWTA ACCCTGGCTG 1201 TTGTCACCAT GG=TATGAA GAACAGAACA GAAATGTAGC MCTGAGACA 1251 GAGGCCAAW AGAAAAT= TCAGGAAWC CAGCAGCTGT TAAWGAGGA 1301 GAAWAGGCT CTGGTTGCCA TGWAATTGA CAGAAGT= CTTAATTCCC 1351 TTCAAW= ATC==C WGAAGAAGA GGAA== CWTAGTAAG 1401 ACAAGAAMT CCTTCTTTAT GAGAGGG= AAGAWG= AAW=AGC 1451 =TGATTCA GAGGACGATG CC=AAAAA TCCACAG= CTTGAGCAGA 1501 CCAAAWACT GTCCCAGAAC TTGWAGTGG ATC=TTGA TGAWAWTG 1551 GACCCCCTCC ACAGWAGAG AGCWTGAGC GCTGTCAGTA TCTTAACCAT 1601 CACCATWAG GAACAAGAAA AATTWAGGA GCCTTGTTTC CCATGTGGGA 1651 AAAATTMGC CTCTAAGTAC CTGGTGTGGG ACTGTAGCCC TCAGTGWM 1701 TWATAAAGA AGGTCCTGCG GACCATCATG ACGGATCCCT TTACTGAWT 1751 GGWATCACC ATCMCATCA TCATCAATAC CGTTTTCTTA GCCGTGGAGC 1801 ACCACAACAT GGATGACAAC =AAAGACCA TACTGAAAAT AGGAAA=G 1851 GTTTTCACGG GAATT=CAT AGWGAAAM M=CAAGA TCATCGCWT 1901 CGACCCTTAC CACTACT= WCACG=G GAATWT= GACAWATCG 1951 TGWCCT= GAGTCTCGCT GAMT=CT ACAACACACT G=GATAAC 2001 AATAGGT= TCTTGGCTTC CCTCAGAGTG C7PGAGGG= TCAAGTTAGC 2051 CAAATCCTGG WCAWTTAA ACACT=AT TAAGATCATC GGCCACT= 2101 TGGGCGCGCT I.GGAAACCTG ACTGTGG= TGACTATCW GGT=CATC 2151 T==TGG TGGGCATGCG G==GGC ACCAAGT= ACAAGAWGC 2201 CTACWCACC CAGGAWGGC CCAGGWGW CTGWACATG GATAATTTCT 2251 ACCACTC= CCTGGTGGTG TTCCGCATCC TCTG'"GGGGA ATGGAIPWAG 2301 AACATGTGGG GCTGCATGCA GGATATGGAC GGCTCCCCGT TGTGCATCAT 2351 TGTCTTTGTC CTGAMATGG TGATCWGAA G=GTGWG CTMACC= 48 1 1 2401 TCATTGW= 2451 CTGGAAGGAG 2501 CCGCCGGGCC 2551 AGAAATWAG 2601 TTTGCTGGTG 2651 GGAGTATGAT 2701 TGWWCACT 2751 GGMCCCTAC 2801 CCCTCCAGAG 2851 TGGAAGTATT 2901 AAGAAGT= 2951 GAATGATATC 3001 CAGATAGATG 3051 ACAGACAAGA 3101 CTGCTACCAA 3151 TTATTCT= 3201 AGCCGGCCCC 3251 ATTTATT= 3301 GGAGGTATTT 2251 GTGTCTGTGC 3401 TCTGCGGGCC 3451 AGGTTGT= 3501 TTGWGG= 3551 TTTATTTTCT 3601 TGTATTTGGA 3651 AATTACT= 3701 CTATW=C 3751 TGAATGWGC GCTGCTCAAT AGACCAGGAA TTCTCCTTCA GAGGAAAAAC AGAATAAAGA ACAGACATGG WCAGAGGTA CCACCTCACA ACCAAWAGC T=GAAGAA ACWAGTGAG TTTAGAAATT CTTTCCCAAG GAAAGTCCCC ATCGTGAAGC GAWAGTGGA AAGTTGAGAA CTCCTGGAAA CACCAGMCC TCAG=CAT CMAGACCTC CTACWC= GCCTCATTTT GGGAAGTTTG TTTTACCGAA GGAWGT= TC=CAWA AACCAAAGTG TGCTGCACGC TWCCAAAW WCAATCCTC CTTTGTACAC GAGGACGATG ACATAGT= TCACTAG= GATCMCATT CATGCTCTCG TACAGAAAAC GG=TAG= CTGGGTCCTG ACAGCTGGTT GCWTGATAT ATTACTAAGG TGATCCTGAA TGW=GGC GAATCTACCA TGWGGCWT ATCAGWCCA CTGGCTCGTA GAAGGMCAT GTTCCGAACC WAGGTCAAC CTGCTGCAAG TGWAACCTA T=GATTCC AGAGAGAAAG 49 ATGAGGAGAA CAGCTAG= T=CAGAGT CAAAAGAGAC CWGATGWA TWACAGGCC TWAATATTG GGAWTCAGG GGATGACCAA TAMCATACA GAATWAGCA AGT==C GTCACTT= TGWGGAMA TGAGAGT= TTGAAGAMT TGTACCGATA GTGWTGWC TTGATTT= AWTTGAAGT GTCCCAGTTT TACCTGWAT TTTTGTATCT MACWGACA G-kAGCCAA-TG TTTGACAAW MAGGG=G AWAWAGCC GGATGWAGC TGGATCGGTT TTTTGTTGCA AACAGAAAW GGCCCTGGAA GGWC=GC M=AAGGC CCGGTGACCT GGGGTTGAAA GAG=TWA CAATTGACCT AAAAAWAGC ATGWACAAA TTCGGAAAAC ATAATC=G CAATWC= ATATT=CAC TTTGGATTCC CATT== CWT=GAC GAAGGAATGA TCTCAATGTC MGGAGTAAA GACATAAATA TAACATTAGT TGGGGAATGC WGGAA.ATCA GGACTTMAG 3801 GCGAACCTCT ACWGTATCT CTACTT=G G=TTATCA TCTTCGGCTC 3851 CTTCTTTACC CTGAACCTCT TTATCGG= TATTATTGAC AAWTCAATC 3901 AWAWAGAA AAAGTTAGGT GWCAAGACA TCTT-CATGAC TGACzGA-GCAG 3951 AAGAAATATT ACAATWAAT GAAAAAWTA GGAMCAAGA AAWTCAAAA 4001 GCCCATCCCA AGG=CMA ACAAATGTCA AGCCTTTGTG =WACCTGG 4051TCACAAWCA GG=TTGAC GTCATCATTC TGG=TTAT TGTCTTAAAT 4101 ATGATTATCATGATGWTGA ATCTGWGAC CAGWCAAAG ATWGAAGAA 4151 AACWTTGAT ATCCTCAACA TAGCCTTCGT GGTCATC= ACCATAGAGT 4201 G=CATCAA AG=TTGW TTGAGGCAAC ACTACTTCAC CAATGWMG 4251 AACTTAT= ATTGMTGW CGTGGTTCTT TWATCATTA GTACWTGW 4301 TTCCCGCTTG GAGGACAGTG ACATT=TT CCCGCCCACG CTCTTCAGAG 4351 TCGTCCGCTT GG=GGATT GGTCGAATCC TCAGGWWT CCGGGCTGCC 4401 CGGGGAATCA GGACCCTCCT CTTTGCTTTG ATGATGTCTC TWC-TC= 4451 =CAACATC G=TGWGC TCTTCCTGGT GAT=CATT TACWCATCT 4501 TTGWATGAG CTGGTTTTCC AAAWGAAGA AGGG=CW GATWAWAC 4551 ATC=CAACT TCGAGACCTT TAWGWAGC ATGWGTGW TCTTCCAGAT 4601 AACCACT= GCTGGCTGGG ATACCCTCCT CAAWCCAM CTGGAGGCAA 4651 AAGAMACTG CAA==C =CAAGACA GCTGTCAGCA GCWCAGATA 4701 GCCGTCGTCT ACT=TCAG TTACATCATC ATCTCCTTCC TCATC=W 4751 CAACATGTAC ATCWTWGA TC=GAGAA CTTCAACACA GCCACGGAGG 4801 AGAGWAGGA CCCTCTGGGA GAGGAWACT TTGAAATC= CTATGAGG= 4851 TGWAGAAW TTGACCWGA GGCGTCGCAG TTCATCCAGT ATTCWC= 4901 CTCTGACTTT GCGGACGCCC MCCGGAGCC GTTGW=G WCAAGWGA 4951 ATAAWTTCA GT=TAGM ATWACTTGC CCATGWGAT GGGWACCW 5001 CTCCATTGCA TGGATGTTCT CTTTGCTTTC AC7PACCAGGG TC=GWGA 5051 CTCCAGCGGC TTGGATACCA TGAAAAWAT GATGGAGGAG AAGTTTATGG 5101 AGGWAMCC =TTAAGAAG CTCTACGAGC CCATAGTCAC CACCACCAAG 5151 AGGAAWAGG AGGAGCAAGG CGCCGCCGTC ATWAGAGGG CCTACCGGAA 50 1 5201 ACACATGGAG AAGATWTCA AACTGAGGCT GAAWACAGG TCAA=CAT 5251 WCACCAGGT G=MCA.AT GGAGACT= CCAWTTGGA TGTGGCCAAG 5301 GTCAAGGTTC ACAATGAC= &ACCCTCATC TAGA

Claims (1)

1. 2.

   3.

   4.

   5. 6. 7. 8. 9 10.

   12. 13. 14.

   What is claimed is:

   1. An isolated DNA sequence comprising the nucleotide sequence set forth in SEQ ID NO: 1 or SEQ ED NO:3. The DNA of Claim I wherein said DNA sequence is encoding a sodium channel protein ZP or fragment thereof. The DNA of Claim 2 wherein said sodium channel protein is the (x-subunlt or fragment thereof. The DNA of Claim 3 wherein said sodium channel protein is tetrodotoxin-resistant. The DNA of Claim 3 or 4 wherein said sodium channel protein is found in mammals. The DNA of Claim 3 or 4 wherein said sodium channel protein is found in rat. The DNA of Claim 3 or 4 wherein said sodium channel protein is found in human. The DNA of Claim I wherein said DNA is cDNA. The DNA of Claim 1 wherein said DNA is synthetic DNA. Expression vectors comprising the DNA of Claim 8. Expression vectors comprising the synthetic DNA of Claim 9. Host cells transformed with the expression vectors of Claim 10. Host cells transformed with the expression vectors of Claim 11. A recombinant polynucleotide comprising a nucleic acid sequence derived from the DNA sequence of Claim 1. A sodium channel protein encoded by a DNA of Claims 1 to 9 or allelic variants thereof. A tetrodotoxin-resistant sodium channel protein encoded by a DNA of Claims 1 to 9 or allehc variants thereof. The protein of Claim 16 having the amino acid sequence set forth in SEQ ID NO:2. 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. Poly- and/or monoclonal antibodies raised against a tetrodotoxin-resistant sodium channel protein encoded by a DNA of Claims I to 9 or allelic variants thereof. A diagnostic kit comprising a polynucleotide of claim 14 capable of specifically hybridizing to a tetrodotoxin -resistant sodium channel protein or fragment thereof.

   r C The use of an isolated DNA sequence of Claims I to 9 for identifying a compound suspected of being an inhibitor of tetrodotoxin-resistant sodium channel protein. The invention substantially as hereinbefore described especially with reference to the foregoing Examples.

   Z) C) 15. 16.

   17. 18.

   19.

   20.

   21.

   22.

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