AU9327298A - Nucleic acid encoding a nervous tissue sodium channel - Google Patents

Description

S F Ref: 441828

AUSTRALIA

PATENTS ACT 1990 COMPLETE SPECIFICATION FOR A STANDARD PATENT

ORIGINAL

Name-- an Adrs Name and Address of Applicant: Actual Inventor(s):

'U

t I F. Hoffmann-La Roche AG 124 Grenzacherstrasse CH-4070 Basle

SHITZERLAND

Paul Shartzer Dietrich, Douglas Kenneth Rabert, Linda Marie Fish, Reena Khare, Lakshmi Sangameswaran

II

0 6

S

0O S r rs Address for Service: Invention Title: Spruson Ferguson, Patent Attorneys Level 33 St Martins Tower, 31 Market Street Sydney, New South Hales, 2000, Australia Nucleic Acid Encoding a Nervous Tissue Sodium Channel The following statement is a full description of this invention, including the best method of performing it known to me/us:- 5845 1 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 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 essential for the generation of 5, action potentials in excitable cells. An excellent review of sodium channels is presented in 15 Catterall, TINS 16(12), 500-506 (1993).

The cDNAs for several Na channels have been cloned and sequenced. Numa et al., S' 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, 20 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 ,25 channels include rat brain types I and II, Noda et al., Nature 320, 188-192 (1986), Ha, Auld et al., Neuron 1, 449-461 (1988), and III, Kayano et al., FEBS Lett. 228, 187-194 (1988), rat skeletal muscle (SkM1), 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., 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, 30125- 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 I- 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 ,,isoform" is o°0• 0 used to mean distinct but closely related sodium channel proteins, 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 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 IC 5 0 values: TTX-sensitive Na channels (IC 5 0 1-30 nM), TTX-insensitive Na channels (IC 5 0 1-5 gM), and TTX-resistant Na channels

(IC

5 o 2 50 gM).

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 (IC 5 o 0.3 nM) and TTXresistant (IC 5 0 100 gM) 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 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. 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.

gg 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.

25 Further provided is a method of inhibiting the activity of the TTX-resistant sodium c channel comprising administering an effective amount of a compound having an IC 5 0 of [VM 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 SEO ID'S AND FIGURES Figures 1A-E depict the 5908 nucleotide cDNA native sequence encoding the rat sodium channel type 5 (SEQ ID NO: 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 cAMP-dependent protein kinase A (PKA) phosphorylation site 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 Figure 4 depicts the sequence for the novel sodium channel domain IV probe (SEQ ID NO: 4).

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

Figure 6 depicts the cloning map of o* 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 preferably less than about 10%, and even more preferably less than about 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 S nucleotide sequences set forth in Figures 1A-E (SEQ ID NO: 1) designated herein as sodium S* e 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 25 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: This 5334 nucleotide SaII-XbaI 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 E. Coli.

The nucleotide sequence of SEQ ID 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 rPN1, with 72% and rat brain types I and III, with 71.8% and 71.3% respectively. Homology to rPN3a, hPN3, 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 sequence spans the region between IIIS1 and interdomain III/IV which includes the fast inactivation gate IFM) that is located within interdomain II/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

U,

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 voltage-gated, TTX-resistant 25 sodium channel. Shown in Figures 2G-H are the homologous domains the putative transmembrane segments (S1-S6); the amino acid conferring resistance to TTX Nglycosylation sites and cAMP-dependent PKA phosphorylation sites 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 PN1 and PN4, this amino acid is tyrosine or phenylalanine and these channels are all TTX-sensitive. In PN3 and the amino acid is a serine. Since PN3 is highly resistant to TTX, the implication is that is also a TTX-resistant channel. The cardiac channel has a cysteine at this position and is ,,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 thansix 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 DIII 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 RT- PCR 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)

25 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.

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 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.

S o* 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 25 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 ca-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 15 correspond to, 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, N18TG, from Massachusetts General Hospital.

Fl 1 responds to trophic agents, such as NGF, by extending dendrites. It was found that was present in both native F11 and F11 treated with NGF, leading the applicants to believe O that the sodium channel is natively expressed in F11.

25 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.

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

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 a-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 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 B 1 -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 B1-subunit with the a-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, 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 25 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.

S* 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 25 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.

12 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 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.

Biochemistry 18, 5294-5299, (1979)). The RNA is size-fractionated on a sucrose gradient to enrich for large mRNAs because the sodium channel ca-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 S*0 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 MRF' bacteria 25 (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 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 Milstein 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 25 a carrier. 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 cc-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 S° ganglia of the nervous system.

oo 25 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 CI I I 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, reduce the conduction of nerve impulses, will affect the nerve's ability to 25 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 IC 5 0 approximately 10 pLM or less, preferably <1 M. Potential therapeutic compounds are identified based on their ability to inhibit the activity of Therefore, the aforementioned assay can be used to identify compounds having a therapeutically effective IC 5 0 The term ,,IC 50 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).

25 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, Batki, Hynds, and Kricka, 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.

6 6 6 25 6@6@ 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 MOPS: 3 -(N-morpholino)propanesulfonic acid (Sigma Chemical Company) PN5: peripheral nerve sodium channel PNS: peripheral nervous system SDS: sodium dodecyl sulfate SSC: 150 mM NaC1, 15 mM sodium citrate, pH SSPE: 80 mM NaCi, 10 mM sodium phosphate, 1 mM ethylenediaminetetraacetate, pH TEV: two electrode voltage clamp TTX: tetrodotoxin (Sigma Chemical Company) too*.

*9.

EXAMPLES

The following Examples illustrate practice of the invention.

Materials The plasmid pBK-CMV was obtained from Stratagene (La Jolla, CA); the plasmid 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 pCRII 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 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 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, 156- 159 (1987)). Total RNA (5 gg of each sample) was dissolved in MEN buffer containing formamide, 6.6% formaldehyde and denatured at 65 0 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.

S 25 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 gg/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 0 C for 1 hour.

EXAMPLE 2 PROBE FROM RAT BRAIN IIA A 32P-labeled cRNA probe complementary to nucleotides 4637-5868 of the rat brain IIA sodium channel a-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 BstEII.

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 IIA 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 42 0 C. The membrane was hybridized in formamide, 5xSSC, 50 mM sodium phosphate, pH 7.1, lx Denhardt's solution, 0.5% SDS, and sheared, heat-denatured salmon sperm DNA (200 utg/ml) with the 32P-labeled cRNA probe (ca.

1-3x10 6 cpm/ml) described in Example 2 for 18 hours at 42 0

C.

25 The membrane was rinsed with 2xSSC, 0.1% SDS at room temperature for 20 min. and S0 I 0 Sa Sthen washed sequentially with: 2xSSC, SDS at 55 0 C for 30 min., 0.2xSSC, 0.1% SDS at 65 0 C for 30 min., 0.2xSSC, 0.1% SDS at 70 0 C for 30 min., and 0.2xSSC, 0.1% SDS, 0.1% sodium pyrophosphate at 70 0 C for 20 min. The filter was exposed against Kodak X-omat AR film at -80 0 C with intensifying screens for up to 2 weeks.

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

EXAMPLE 4 NOVEL SODIUM CH{ANNEL DOMAIN IV 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 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 novel domain IV sequence was as follows (SEQ lID NO: 4): 0 0* 00 *0 0# 1 51 101 151 201 251 301 351 401 451 501 551 25 601 651 701

CTCAACATGG

GACGAAGGTT

GCGAGTGTGT

GGCTGGAACG

GCTGTTTCT

TCTTCCGGGT

CGAGCAGCCA

GCCCGCCCTC

ACTCCATCTT

ATCGACGACA

TTACGATGAT GGTGGAGACC CTGGGCAGAA TCAACCAGTT GATGAAGATG TTCGCCCTGC TGTTCGACTT CATAGTGGTG GCA.ATCCTTA AGTCACTGGA CATCCGTCTG GCCAGGATCG AGGGGATTCG CACGCTGCTC TTCAACATCG GCCTCCTCCT CGGCATGGCC AGCTTCGCTA TGTTCAACTT CAAGACCTTT

GACGAGCAGG

CTTTGTGGCC

GACAGTACTA

ATCCTGTCCA

AAACTACTTC

GCCGCATCCT

TTCGCCCTCA

CTTCCTCGTC

ACGTCGTGGA

GGCAACAGCA

CGGCCTCCTC

ACCTGCCCAA

GGCATCATCT

CAACATGTAT

GCGAGGAGAA

GTCTTCACGG

TTTCACCAAC

TTGGGAGTCT

TCCCCGACGC

CAGGCTGATC

TGATGTCCCT

ATGTTCATCT

CGAGGCCGGC

TGCTGTGCCT

AGCCCCATCC

CAGCAACGGC

TCTTCACCAC

ATCGCAGTCA

GTTCCAGATC ACCACCTCGG CCGGCTGGGA TCAACACGGG GCCTCCCTAC TGCGACCCCA TCCCGGGGGA ACTGCGGGAG CCCGGCGGTG CTACATCATC ATCTCCTTCC TCATCGTGGT

TC

This sequence was labeled with 32 P by random priming.

EXAMPLE HYBRIDIZATION OF RNA WITH THE NOVEL SODIUM CHANNEL 3'-UTR PROBE A Northern blot was prepared with 10g 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 0 C in a solution containing 5XSSC, 1X Denhardt's solution, 0.5% SDS, 50mM sodium phosphate, pH 7.1, salmon sperm DNA (1mg/ml) and 50% formamide. Hybridization was conducted at 45 0 C for 18 hours in the above solution except that the salmon sperm DNA was included at a concentration of 200gg/ml and the 3 2 P-labeled probe was added at 7.5x105 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 0 C for 20 min., and once with 0.2XSSC, 0.1% SDS and 0.1% sodium pyrophosphate at 65 0 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 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 00* 0 An EcoRI-adapted cDNA library was prepared from normal adult male Sprague- Dawley rat DRG poly(A)+ RNA using the SuperScript Choice System (GIBCO BRL). cDNA kb) was selected by sucrose gradient fractionation as described by Kieffer, Gene 109, 115- 119 (1991). The cDNA was then ligated into the Zap Express vector (Stratagene), and packaged with the Gigapack II XL lambda packaging extract (Stratagene). Similarly, a >2kb DRG cDNA library was synthesized.

Phage (3.5x105) were screened by filter hybridization with a 32P-labeled probe (rBIIa, bases 4637-5868 as follows of Auld et al., 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 0 C and washed in SSC/0.1% SDS at 50 0

C.

Southern blots of EcoRI-digested plasmids were hybridized with the 32P-labeled DNA probe, (SEQ ID NO: The filters were then hybridized in 50% formamide, 6X SSC, Denhardt's solution, SDS, and 100 gg/ml sheared, denatured salmon sperm DNA at 42 0 C and were washed in 0.1X SSC/0.1% SDS at 65 0

C.

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 II to the 3'-UTR. However, each had a deletion which truncated the coding region.

Clone 1.18 had the untranslated region, in addition to the C-terminus of the deduced amino acid sequence of PN5. The construct in the expression vector, pBSTACIIr, 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: 0 *0 *r 0 $0.0

S

Channel PN3a hPN3 PN4 PN4a Similarity 71 71 71 71 Identity 54 53 53 PN1 72 rat brain type I 72 rat brain type II 71 54 rat brain type III 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, pBSTAcIIr, and other vectors) could not be accomplished without use of special growth media, conditions, and E. coli strains. The following proved to be optimal: use of E. coli STBL2TM for primary transformation following ligation reactions and for large scale culturing; solid media was 1/2x FM (see below) plus lx LB (Tryptone, Yeast Extract, NaCI, plus 15g/L agar, or IxFM plus 1/2x LB; liquid media optimally was lx FM plus 1/2x LB; carbenicillin, 100g/ml, was used for all media, as it is metabolized less rapidly than ampicillin; temperature for growth should be no greater than 30 0 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 25 (NH4)2S04 1.8g KH2PO4 3.6g Glycerol 88g qs to IL S 2x FM and the 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 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, Schleif, R.F. and Wensink, 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 pBSTAcIIr 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 Gene 27, 151-160(1984). pBSTAcIIr was digested with PvuI. The 755 bp fragment containing the T7 promoter, B-globin the multiple cloning site, B-globin 3'UTR, and T3 promoter was ligated to the 3.6 kb fragment containing the replication origin, ampicillin resistance gene, rmBT 1 and rrBT 1

T

2 transcription terminators from pKK232-8, which had been fully digested with Smal and partially digested with Pvull and treated with shrimp intestinal phosphatase to prevent self ligation. The resulting plasmid in which the orientation of the pBSTA fragment is suchthat the T7 promoter is proximal to the rmBT 1 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 rrB T 1 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, pBK- CMV. 1.18 could be used to ,,repair" pBK-CMV.26.2. A strategy was developed to assemble a 25 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 SalI 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 XbaI 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 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 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: 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 (GGGTTCAATGTGGTTTTATCT, corresponding to bp 1067 to 1047 of SEQ ID NO: followed by gel purification, digestion with SalI and KpnI (KpnI site at pb 1003-1008, SEQ ID NO: and gel purification.

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

The 3' end fragment was prepared as follows: PCR using primers 4837 25 (TCTGGGAAGTTTGGAAG, corresponding to bp 3613 to 3629 of SEQ ID 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, corresponding to bp 4218 to 4239 of SEQ ID NO: 1) and 4929 (GTCTTCTAGATGAGGGTTCAGTCATTGTG, XbaI site underlined, 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 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 AatII and XbaI, and the 1.2 kb fragment gel purified.

The 3' end fragment was cloned into AatII and XbaI digested pBSTAcIIr. One isloate was digested with Sall 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 pBSTAcIIr.PN5(clone 21). pBSTAcIIr.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 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 NheI (bp 2538-2543 SEQ ID NO: 1) and XhoI (bp 4828- 4833, SEQ ID NO: 1) to give a 6.2 kb fragment and with AatII and XhoI to give a 0.7 kb fragment which were ligated to the 1.6 kp fragment resulting from digestion of pBK- 25 CMV.26.2 with AatII and NheI. 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 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 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 Ile Phe Met Thr Glu Glu Gin 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.

(clone 21)) and the region near the XhoI 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 (GCTCCTCAGTCATGAAGATGTCTTGGCCACCTAAC, correspoind to bp 4003 to 3969, SEQ ID NO: 1, mutated bases are underlined) to give a 320 bp product. Primer P5-4017S (GGCCAAGACATCTTCATGACTGAGGAGCAGAAGAAATATTAC, corresponding to bp 3976 to 4017, SEQ ID NO: 1; mutated bases are underlined) was paired with primer 4247AS (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 4247AS to give a 597 bp product, which was T/A cloned into vector pCRII. Isolate HO-7 was found to have the desired sequence. A four-way ligation was performed to assemble the full- S 25 length, modified 2 1 2 It **2 3 t the oocyte expression vector HQ-8 ws digested with SalI and XbaI 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 half of PN5; HO-7 was digested with Mlul (bp 3866 to 3871, SEQ ID NO: 1) and AatII to give a 0.3 kb fragment containing the mutant 8 bp repeat region of PN5; GPII-1 was digested with AatII 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 is shown in Figures 5A-E (SEQ ID NO: 0 EXAMPLE 8 HUMAN 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 IIIS 1 and interdomain III/IV which includes the fast inactivation gate IFM) that is located within interdomain III/IV.

0 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 II 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, 5 rat PN1 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.

0 Channel Human Brain (HBA) Human Heart (hHl) Similarity 76 81 Identity 69 74 Human Atypical Heart Human Skeletal Muscle Human Neuroendocrine Human PN3 Rat PN1 Rat PN3 Rat PN4 Rat Figure 3B compares the amino acid sequence of the hPN5 fragment with the rat amino acid sequence in the appropriate region.

EXAMPLE 9 TISSUE DISTRIBUTION BY RT-PCR Brain, spinal 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 0 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: 940C/2 min. (denaturation), 94 0 C/30 sec., 65 0 C/30 sec. and 72 0 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).

30 Tissue distribution profile of rPN5 by analysis of RNA from selected rat tissues by RT- PCR was as follows:

C

C

C. e

C

CC.**

C Tissue Brain RT-PCR (35 cycles) Spinal cord DRG Nodose ganglia Superior cervical ganglia Sciatic nerve Heart Skeletal muscle F11-untreated F11-treated PN5 was also detected after only 25 cycles (24 1) in the same five tissues as above in the same relative abundance.

EXAMPLE

ANTIBODIES

A synthetic peptide (26 amino acids in interdomain II and In residues 977 to 1002) was conjugated to KLH and antibody raised in rabbits. The antiserum was subsequently affinity purified.

constitutes a subfamily of novel sodium channel genes; these genes are different from those detectable with other probes 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.

0* 4. e'i SEQUENCE LISTING 1) GENERAL INFORMATION:

APPLICANT:

NAME: F. HOFFMANN-LA ROCHE AG STREET: Grenzacherstrasse 124 CITY: Basle

STATE:BS

COUNTRY: Switzerland POSTAL CODE (ZIP): CH-4010 TELEPHONE: 061-6884256 TELEFAX: 061-6881395 TELEX: 962292/965542 hlr ch (ii) TITLE OF INVENTION: Nucleic Acid Encoding a Nervous Tissue Sodium Channel (iii) NUMBER OF SEQUENCES: (iv) COMPUTER READABLE FORM: MEDIUM TYPE: Floppy disk COMPUTER: IBM PC compatible OPERATING SYSTEM: PC-DOS/MS-DOS SOFTWARE: PatentIn Release 1.0, Version 1.30 INFORMATION FOR SEQ ID NO:1: SEQUENCE CHARACTERISTICS: LENGTH: 5908 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA oe: (iii) HYPOTHETICAL:

NO

(iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: rat TISSUE TYPE: Dorsal root ganglia CELL TYPE: Peripheral nerve (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1: 1 GAAGTCACAG GAGTGTCTGT CAGCGAGAGG AAGAAGGGAG AGTTTACTGA 51 GTGTCTTCTG CCCCTCCTCA GGGTGAAGAT GGAGGAGAGG TACTACCCGG 33 101 151 201 251 301 351 401 451 501 551 601 651 701 751 801 851 901 951 1001 1051 1101 1151 1201 1251 1301 1351 1401

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 AATTTCCGCC AGAAGCGGAT TGCTATCCAA GCAGCTGAGC CCCAGCCTCG GTTACCTAAG CTTTATGGTG TGGAAGACCT GGACCCATTC AACAAGAAGA GAACAATTTA TCTGGGGCCT TTTAATCCCC ATTCAGTCTT TAGCATGTTC TTCATGGCGA ATTCTATGGA CGTCTTCATT GGGATTTATA GAGGCTTCAT TGTGGATGAG CTGGACTTCA TTGTCATTGG CCAAGTCAAT CTTTCAGCTC AGGCGATTTC AGTTATCTCA CGCTCGGTGA AGAAGCTGGT

CCTTCACTTC

AAGGAGAGGA

GCCTCAGCTT

ACATTCCCCC

TACAAAGACC

TCGCTTCAGC

TCAGAAGCTT

ATCATCTGCA

GAGAAGTTTC

TTTTAGAAGC

TTTTCCTTCC

AACAGCGATC

TTCGTACCTT

GGTCTGA.AGG

AGACGTGATG

GTCAGCAGCT

TGTGGCCCCA

TAGCGAAGAC

CCAATGGTTC

ACAAAGTTTG

GACTCAAGAC

GGATCTACTT

TACCTGCTTA

GAACAGAAAT

CGACTCTCTG

AGAAGTCCAA

GACCTAAAGG

TGAGCTTGTA

ATAAGACATT

GCCAAGCGGG

AATGATTCGT

CGGTGATCAT

GACAACGACA

TGTGATTAAA

TCCGAGATCC

GCAACTTGTT

CCGAGTGTTC

TCATCGTAGG

GTCCTCACTC

GTTCATGGGA

ACCCTGCATC

TTCATAATGT

TACGTGCGAT

ACAACTTTGG

TCCTGGGAGA

TGTCTTCTTC

AC CTAAC CCT

GTAGCTGCTG

GCTGTTAAGG

GTTCCCTTAA

TTTTTCGGTA

0S a 0~ 0 a 0 *000 a

CAGCATCTTT

AGAAGTGTAT

TGCTTTGAAA

GCTCGGCAGC

TGAACCCAGA

CTCGCCATGT

ACAGATCCTG

TCATCTTCCT

ACCATGGCTT

CAAGGAGAAA

AGGCTCTGGT

GCTTCATCCT

GCCCTGGTCG

TAAGCACAAC

AGGAAAAAGA

AGACCCTGTC

CAATAATTAT

TCCGGGTTAT

CGGACCTCTG

GGGCTCCTTC

ATGAAGAACA

ATGTTTCAGG AAGCCCAGCA TGCCATGGGA ATTGACAGAA TTTCCCCGAA GAAGAGGAAG 34

I

1451 1501 1551 1601 1651 1701 1751 1801 1851 1901 1951 2001 2051 2101 2151 2201 2251 2301 2351 2401 l6 oo 2451 2501 2551 2601 2651 t 2701 2751 2801

GTAAGACAAG

TCAGCGTCTG

GCAGACCAAA

ACGTGGACCC

ACCATCACCA

TGGGAAAAAT

GGCTGTGCAT

GAGCTGGCCA

GGAGCACCAC

ACTGGGTTTT

GCGCTCGACC

CATCGTGGCC

ATAACAATAG

TTAGCCAAAT

CTCCGTGGGC

TCATCTTTTC

ACCGCCTACG

TTTCTACCAC

TCGAGAACAT

ATCATTGTCT

CCTCTTCATT

GGAGCCTGGA

CGGTTCCGCC

TTGCAAGAAA

AAAGCTTTGC

TGGAAGGAGT

TCCGCTGGCC

AAGGCGGTGC

AAAGTCCTTC

ATTCAGAGGA

CGACTGTCCC

CCTCCACAGG

TGCAGGAACA

TTGGCCTCTA

AAAGAAGGTC

TCACCATCTG

AACATGGATG

CACGGGAATT

CTTACCACTA

CTCCTGAGTC

GTCTTTCTTG

CCTGGCCCAC

GCGCTTGGAA

TGTGGTGGGC

CCACCCAGGA

TCCTTCCTGG

GTGGGGCTGC

TTGTCCTGAT

GCCTTGCTGC

AGGAGAGACC

GGGCCTTCTC

TGCAGGAGGA

TGGTGAGAAT

ATGATACAGA

CCACTCGCAG

CCTACCCACC

TTTATGAGAG

CGATGCCTCT

AGAACTTGCC

CAGAGAGCGC

AGAAAAATTC

AGTACCTGGT

CTGCGGACCA

CATCATCATC

ACAACTTAAA

TTCATAGCGG

CTTCCGGCAC

TCGCTGATGT

GCTTCCCTCA

GTTAAACACT

ACCTGACTGT

ATGCGGCTCT

GCGGCCCAGG

TGGTGTTCCG

ATGCAGGATA

AATGGTGATC

TCAATTCCTT

AGGAAAACCA

CTTCATGCTG

AAAACTCGCC

AAAGACTCAA

CATGGCTTTG

AGGTAGAGGA

TCACAACATA

GGTCCAAGAC GGCCCAAGCC AAAAATCCAC AGCTCCTTGA AGTGGATCTC TTTGATGAGC TGAGCGCTGT CAGTATCTTA CAGGAGCCTT GTTTCCCATG GTGGGACTGT AGCCCTCAGT TCATGACGGA TCCCTTTACT AATACCGTTT TCTTAGCCGT GACCATACTG AAAATAGGAA AAATGTGTCT CAAGATCATC GGCTGGAATG TTTTTGACAG GCTCTACAAC ACACTGTCTG GAGTGCTGAG GGTCTTCAAG CTCATTAAGA TCATCGGCCA GGTCCTGACT ATCGTGGTCT TCGGCACCAA GTTTAACAAG CGGCGCTGGC ACATGGATAA CATCCTCTGT GGGGAATGGA TGGACGGCTC CCCGTTGTGC GGGAAGCTTG TGGTGCTTA CAGCAATGAG GAGAAGGATG AAGTGCAGCT AGCCCTGGAT CACGCTCTTC AGAGTTTTTG AAAGCCAAAA GAGACAACAG TCCTCCCGGA TGCGAGGCCC TACACTGGAC AGGCCGGGGC CGATGTGGAA TATTGTGGTG GTGCTGGAGT TCAGGCCGGT 2851 GACCTCCCTC CAGAGACCAA GCAGCTCACT AGCCCGGATG

ACCAAGGGGT

2901 2951 3001 3051 3101 3151 3201 3251 3301 3351 3401 3451 3501 3551 3601 3651 3701 3751 3801 3851 3901 3951 4001 4051 4101 4151 TGAAATGGA7 CTCGAAAGA7Z

GACCTGAATC

GCAGCCAGA1

ACAAAACAG.P

AAAACCTGC'I

CTTTGTTAT'I

TCCCCAGCCG

TTCACATTTA

ATTCCGGAGG

TGGTGGTGTC

CGGACTCTGC

AATGAAGGTT

ATGTCTTGCT

GTAAATTTAT

AAATATGTAT

TTAGTAATTA

AATGCCTATC

AATCATGAAT

TTGAGGCGAA

GGCTCCTTCT

CAATCAGCAG

AACAGAAGAA

CAAAAGCCCA

CCTGGTCACA

TAAATATGAT

GTATTTTCTG

GTCTGACGCA

ATATCTTTAG

AGATGCTTTC

CAAGAGAAAG

ACCAAATCGT

CTGCTGAGCA

GCCCCAAGTT

TTTTCCTCCT

TATTTCACCA

TGTGCTCAGT

GGGCCCTGAG

GTCGTCTACG

GGTCTGCCTC

TTTCTGGGAA

TTGGATTTTA

CTCGTGGAAG

TCGCCCTGCT

GCTGCTGTCG

CCTCTACGCG

TTACCCTGAA

CAGAAAAAGT

ATATTACAAT

TCCCA.AGGCC

AGCCAGGTCT

TATCATGATG

AAGAAGATCT

GTGAGCATGC

AAATTTACAG

CCAAGGGCCT

TCCCCCTGGG

GAAGCACAGC

GTGGAGCGCT

GAGAAATTAC

GGAAATGATC

GTGCCTGGTG

CTCATGAATC

ACCTCTGCGG

CCCTGATCAG

ATTTTCTGGC

GTTTGGAAGG

CCGAAGTTCC

GTCCCGCAGG

GCATTTAAGC

TCTCGGAATG

AAAACAGTTT

TAGTTGTCAC

TCCTGTGGTG

TGGTTTGAGA

GATATTTGAA

TAAGGTGTAC

CTGAAGTGGG

CTGGCTTGAT

TACCAAGCTT

GCGCTGTCCC

CGCCATACCT

TCGTATTTTG

TGCATTAACG

GAACCGAAGC

TCAACTTTGA

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

es 0e@*

S

S.

S

S

S

500S S S S. S

S

GCAAGTGGCA

ACCTATAAGG

ATTCCAGAGA

GAAAGACGAG

TATCTCTACT

TTGTGGTTTT

CCTCTTTATC

GGTGTTATTA

TAGGTGGCCA

AGACATTTTT

GCAATGAAAA

AGTTAGGAAC

CCTGAACAAA

TGTCAAGCCT

TTGACGTCAT

CATTCTGGGT

GCTGAATCTG

CCGACCAGCC

36 4201 4251 4301 4351 4401 4451 4501 4551 4601 4651 4701 4751 4801 4851 2 4901 4951 5001 5051 5101 5151 5201 5251 530 5301 540 5401 5501 5551

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

CAACATAGCC

TTGCTTTGAG

GTGGTCGTGG

CAGTGACATT

GGATTGGTCG

CTCCTCTTTG

GCTGCTCTTC

TTTCCAAAGT

ACCTTTACGG

CTGGGATACC

CCTCCTCCCA

GTCAGTTACA

TGTGATCCTC

TGGGAGAGGA

CCCGAGGCGT

CGCCCTGCCG

TAGTGATGGA

GTTCTCTTTG

TACCATGAAA

TTCGTGGTCA

GCAACACTAC

TTCTTTCTAT

TC'PTTCCCGC

AA'rCCTCAGG CT'rTGATGAT

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

ACACAGOCAC

ATCTTCTATG

CCAGTATTCG

GTGTGGCCAA

GTGATGGGCG

CAGGGTCCTC

AGGAGAAGTT

GTCACCACCA

GAGGGCCTAC

ACAGGTCAAG

TTGGATGTGG

CCCTACCTCA

GCAGACTCAC

GTGACAGGTT

GAGAGATGTT

GGAGGACAGT

AACCCTTTTA AGAAGCTCTA OGAGGAGGAG CAAGGCGCCG TGGAGAAGAT GGTCAAACTG CAGGTGTTTT GCAATGGAGA GGTTCACAAT GACTGAACCC GCTTAGCCTC CAGCCTCTGG CCGTTCGATC TGTGTTTTTG TTTAAATGAC TCTTGGAAAG GCAAAGGACA CCGACCATAA 37 5601 5651 5701 5751 5801 5851 5901

CCAACTTACA

CAGATTGTGT

TATGTGACCT

CAGTAAGTTG

GGTGTATGAC

CCGACTTTCA

GGTAAAAG

TAAAGATGAG

TCTCAGTACA

GCCACATGTA

ATAGCTTGCT

TCAAACCTAA

GACGCTCCAA

AAACAAGAAG

TCCCCCAATG

GCTCTTTTTT

ACGGGTGTTC

AAGCATGACT

TCTCTGTCCC

GAAAGATCCC

TGTCTGTTCG

GCATGTACGT

CTACCAGCAT

CTGACTTGTC

AGGTGTCTA-A

AGGAAAACTT

GTGTTTTGAG

CAAAACCCTG

CACAGAATTG

AGTCAGCACC

CGAATAAATA

INFORMATION FOR SEQ ID NO:2: SEQUENCE CHARACTERISTICS: LENGTH: 1765 amino acids TYPE: amino acid

STRANDEDNESS:___

TOPOLOGY: not relevant (ii) MOLECULE TYPE: protein (iii) HYPOTHETICAL: YES (vi) ORIGINAL SOURCE: ORGANISM: rat TISSUE TYPE: dorsal root ganglia CELL TYPE: peripheral nerve (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2: Met Giu Giu Arg Tyr Tyr Pro Vai Ile Phe Pro Asp Glu Arg Asn Phe 9* es..

0S @6 0 0

CO

C

0 S .0 0 so

S

@050 S 0 *5 0 0 Arg Pro Phe Thr Ser 20 Ile Gin Lys Giu Arg 35 Gin Pro Arg Pro Gin 50 Leu Tyr Giy Asp Ile 65 Leu Asp Pro Phe Tyr Lys Arg Thr Ile Tyr 100 Asp Ser Leu Aia 25 Lys Aia Ile Giu Lys Lys Lys Leu Asp Pro Pro Asp Lys Aia Aia Lys Arg Ile Aia Aia Giu Pro Leu Pro Lys Lys Aia Ser Giu Leu Vai 70 Lys Aia 75 Phe Pro Leu Giu Asp Asp His Lys Met Val Leu Asn Lys Ile Leu Arg Phe Ser Aia 105 38 Arg Aia Leu Phe 110 Gly Pro Phe Asn Pro Leu Arg Ser Leu Met Ile Arg Ile Ser Val His 115 120 125 Ser Phe 145 Tyr Ala Asn Pro Arg 225 Gly Thr Met Pro Phe 305 Ser Phe Gin Ile Tyr 385 Val 130 Met Val Arg Trp Gly 210 Ala Ala Leu Gly Ala 290 Ile Thr Asp Asp Tyr 370 Leu Phe Ala Phe Gly Leu 195 Ser Leu Leu Phe Ile 275 Ser Met Cys Asn Ser 355 Phe Leu Ser Asn Ile Phe 180 Asp Gin Lys Leu Cys 260 Leu Asn Cys Asp Phe 340 Trp Val1 Asn Met Ser Gly 165 Ile Phe Val Al a Arg 245 Leu Asn Lys Gly Lys 325 Gly Glu Phe Leu Phe Met 150 Ile Val Ile Asn Ile 230 Ser Ser Gin Asp Thr 310 Thr Trp Arg Phe Thr 390 Ile 135 Glu Tyr Asp Val Leu 215 Ser Val1 Ile Lys Cys 295 Trp Thr Ser Leu Phe 375 Leu Ile Arg Ile Giu Ile 200 Ser Val Lys Phe Cys 280 Phe Leu Leu Phe Tyr 360 Val Ala Cys Ser Leu Phe 185 Gly Ala Ile Lys Aia 265 Ile Giu Gly Asn Leu 345 Arg Val Val 0* if *0e01.

S@ 0 0* 0 Thr Phe Gi u 170 Ser Thr Leu Ser Leu 250 Leu Lys Lys Ser Pro 330 Aia Gin Val Val Val1 Asp 155 Ala Phe Ala Arg Gly 235 Val1 Val His Glu Arg 315 Asp Met Ile Ile Thr 395 Ile 140 Asn Val Leu Ile Thr 220 Leu Asp Gly Asn Lys 300 Pro Asn Phe Leu Phe 380 Met Ile Asp Ile Arg Ala 205 Phe Lys Val Gin Cys 285 Asp Cys Asn Arg Arg 365 Leu Aa Asn Ile Lys Asp 190 Thr Arg Val Met Gin 270 Gly Ser Pro Tyr Val 350 Thr Gly Tyr Cys Met Pro Giu 160 Ile Leu 175 Pro Trp, Cys Phe Val Phe Ile Val 240 Val Leu 255 Leu Phe Pro Asn Glu Asp Asn Gly 320 Thr Lys 335 Met Thr Ser Giy Ser Phe Giu Glu 400 Gin Asn Arg Asn Val Aia Ala Giu Thr Giu Ala Lys Giu Lys Met Phe 405 410 415 Gin Giu Ala Gin Gin Leu Leu Arg Glu Glu Lys Giu Ala Leu Val Ala Met Ser Phe 465 Asp Ser His Gin Leu 545 Ile Ala His Trp Ala 625 Ser Ser Phe Giy Pro 450 Met Asp Gin Arg Glu 530 Ala Lys Ile His Val 610 Leu Ile Asp.

Lys Ile 435 Lys Arg Ala Asn Gin 515 Gin Ser Lys Thr Asn 595 Phe Asp Vai Asn Leu 675 420 Asp Lys Giy Ser Leu 500 Arg Glu Lys Vai Ile 580 Met Thr Pro Ala Asn 660 Ala Arg Arg Ser Lys 485 Pro Ala Lys Leu 565 Cys Asp Giy Leu 645 Arg Lys Ser Lys Lys 470 Asn Vai Leu Phe Leu 550 Arg Ile Asp I le His 630 Leu Ser Ser Ser Phe 455 Thr Pro Asp Ser Gin 535 Val Thr Ile Asn Phe 615 Tyr Ser Phe Trp Leu 440 Phe Ala Gin Leu Ala 520 Giu Trp Ile Ile Leu 600 Ile Phe Leu Leu Pro 680 425 Asn Gly Gin Leu Phe 505 Vai Pro Asp Met Asn 585 Lys Ala Arg Al a Ala 665 Thr Ser Ser Ala Leu 490 Asp Ser Cys Cys Thr 570 Thr Thr Glu His Asp 650 Ser Leu Leu Lys Ser 475 Glu Giu Ile Phe Ser 555 Asp Val1 Ile Met Gly 635 Val Leu Asn Gin Thr 460 Ala Gin His Leu Pro 540 Pro Pro Phe Leu Cys 620 Trp Leu A.rg Thr Ala 445 Arg Ser Thr Val Thr 525 Cys Gin Phe Leu Lys 605 Leu Asn Tyr Val Leu 685 430 Ser Lys Asp Lys Asp 510 Ile Gly Trp Thr Ala 590 Ile Lys Vai Leu 670 Ile Ser Ser Ser Arg 495 Pro Thr Lys Leu Glu 575 Vai Gly Ile Phe Thr 655 Arg' Lys Phe Phe Giu 480 Leu Leu Met Asn Cys 560 Leu Glu Asn Ile Asp 640 Leu Val Ile S S 5* 0

S

4* 0

S

S S S. S

S

Ile Gly His Ser Val Giy Ala Leu Giy Asn Leu Thr Vai Val Leu Thr Ile 705 Lys Trp Leu Asp Gly 785 Phe Thr Met Asn Lys 865 Asp Ala Pro Glu Val 945 Lys Asn Val Phe His Cys Gly 770 Lys Ser Lys Leu Ser 850 Asp Met Giu Thr Thr 930 Phe Ser Asp Val1 Asn Met Gly 755 Ser Leu Asn Val His 835 Pro Ser Ala Val Ser 915 Lys Ser Asp.

Ile Phe Lys Asp 740 Giu Pro Val Giu Gin 820 Ala Lys Ile Leu Giu 900 Gin Gin Giu Ala Phe Ile Thr 725 Asn Trp Leu Vai Giu 805 Leu Leu Pro Leu 885 Asp His Leu Giu Val 965 Arg Phe 710 Ala Phe Ile Cys Leu 790 Lys Ala Gin Lys Pro 870 Thr Asp Ser Thr Asp 950 Ser Asn Ser Tyr Tlyr Giu Ile 775 Asn Asp Leu Ser Giu 855 Asp Gly Val1 Ala Ser 935 Leu Met Leu Val1 Ala His Asn 760 Ile Leu Gly Asp Phe 840 Thr Ala Gin Giu Gly 920 Pro His Leu Gln Vai Giy Thr Gin 730 Ser Phe 745 Met Trp, Val Phe Phe Ile Ser Leu 810 Arg Phe 825 Cys Cys Thr Glu Arg Pro Ala Giy 890 Tyr Cys 905 Val Gin Asp Asp Leu Ser Ser Giu 970 Lys Thr 985 Met 715 Giu Leu Gly Val Ala 795 Glu Arg Lys Ser Trp 875 Ala Gly Aia Gin Ile 955 Cys Val Arg Arg Val Cys Leu 780 Leu Gly Arg Lys Phe 860 Lys Pro Glu Giy Giy 940 Gin Ser Ser Leu Pro Vai Met 765 Ile Leu Giu Ala Cys 845 Al a Giu Leu Gly Asp 925 Val Ser Thr Pro Phe Arg Phe 750 Gin Met Leu Thr Phe 830 Arg Gly Tyr Al a Gly 910 Leu Giu Pro Ile Lys2 990 Gly Arg 735 Arg Asp Val Asn Arg 815 Ser Arg Giu Asp Pro 895 Ala Pro Met Arg Asp 975 Lys Thr 720 Arg Ile Met Ile Ser 800 Lys Phe Lys Asn Thr 880 Leu Leu Pro Giu Lys 960 Leu Gin 66 S. Sq S 5@ S.

S

6 6*0*5.

6 6* 05 6 ~I'fq I 980 Pro Asp Arg Cys Phe Pro Lys Gly Leu 41 Ser Cys His Phe Leu Cys His 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 1070 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 1170 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 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 Ile 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 Ile Ile Phe Gly Ser Phe Phe Thr Leu 1265 1270 1275 1280 Asn Leu Phe Ile Gly Val Ile Ile Asp Asn Phe Asn Gin Gin Gin Lys 1285 129n 1onc 9 9 0O9*~ .9 9 t tt -L V -3 Lys Leu Gly Gly Gin Asp Ile Phe Met Thr Giu Giu Gin Lys Lys Tyr 1300 Tyr Asn Ala Met 1315 Pro Arg Pro Leu 1330 Ser Gin Val Phe 1345 Ile Ile Met Met Thr Phe Asp Ile Lys Lys Leu Giy 132( Asn Lys Cys Gin 1335 Asp Vai Ile Ile 1350 Ala Giu Ser Ala 1365 Leu Asn Ile Ala 1305 1310 Thr Lys Lys Pro Gin Lys Pro Ile 0 1325 Ala Phe Val Phe Asp Leu Val Thr 1340 Leu Gly Leu Ile Val Leu Asn Met 1355 1360 Asp Gin Pro Lys Asp Val Lys Lys 1370 1375 Phe Val Val Ile Phe Thr Ile Glu 1385 1390 Arg Gin His Tyr Phe Thr Asn Giy 1405 V7al Val Leu Ser Ile Ile Ser Thr 1380 Cys Trp, Ile Lys 1395 Leu Phe Val Asp Phe Ala Leu 1400 Cys Val Val 14AL 1415 1420 Leu Val Ser Arg Leu Giu Asp Ser Asp Ile Ser Phe P 1425 1430 1435 Phe Arg Val Vai Arg Leu Ala Arg Ile Gly Arg Ile L 1445 1450 Arg Ala Ala Arg Gly Ile Arg Thr Leu Leu Phe Aia L 1460 1465 Leu Pro Ser Leu Phe Asn Ile Giy Leu Leu Leu Phe L 1475 1480 1 Ile Tyr Ala Ile Phe Gly Met Ser Trp, Phe Ser Lys V 1490 1495 1500 Ser Giy Ile Asp Asp Ile Phe Asn Phe Giu Thr Phe TI 1505 i510 1515 Leu Cys Leu Phe Gin Ile Thr Thr Ser Ala Gly Trp A~ 1525 1530 Asn Pro Met Leu Giu Ala Lys Giu His Cys Asn Ser SE ~ro Pro Thr Leu 1440 peu Arg Leu Val 1455 eu Met Met Ser 1470 eu Val Met Phe 485 al Lys Lys Giy ar Gly Ser Met 1520 3p Thr Leu Leu 1535 ~r Ser Gin Asp 1550 1 Ser Tyr Ile a Val Ile Leu Leu Gly Giu a 0 00*a S~

C

S

0 00 1540 Ser Cys Gin Gin Pro Gin Ile Ala 1555 Ile Ile Ser Phe 1560 Leu Ile Val Vail 1545 Jai Vai Tyr Phe Va1 ksn Met Tyr Ile Al 1580 ;lu Ser Glu Asp Pr 3 1570 Giu Asn Phe Asn Thr Ala 1575 Thr Glu C 4 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 Gin Phe Ile 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 1685 1690 1695 Phe Lys Lys Leu Tyr Glu Pro Ile Val Thr Thr Thr Lys Arg Lys Glu 1700 1705 1710 Glu Glu Gin Gly Ala Ala Val Ile 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 SGin 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 INFORMATION FOR SEQ ID NO:3: SEQUENCE CHARACTERISTICS: LENGTH: 856 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO S(iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: human TISSUE TYPE: Dorsal root ganglia CELL TYPE: Peripheral nerve (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: 1 GCTGAGCAGT GGGGCACTGA TATTTGAAGA TGTTCACCTT GAGAACCAAC 44 51 CCAAAATCCA AGAATTACTA AATTGTACTG ACATTATTTT TACACATATT 101 151 201 251 301 351 401 451 501 551 601 651 701 751

TTTATCCTGG

TTTCACCAGT

TGACCACCCT

GCACTGAGGC

GGTCAATGCT

TCTGCCTCAT

TCTGGAAAAT

TACCATCATT

TCAACCAGAA

CTGCAAGTGG

TGATTCCACA

GTTACATTTA

AATCTCTTCA

GTTAGGTGGC

ATGCAATGAA

CCCGTT

AGATGGTACT AAAATGGGTA GCCTGGTGCT GCCTTGATTT CATTAACTTA ATGGAATTGA GCCTTCGGAT TTGGAAAGTA

CTCTTCGTGC

CTCATAGGTG

TTTCTGGCTC

TTGGGAAATG

ACAAATAPAA

AGTCAACTTT

CAACATTTAA

GAGAAAGAAC

CTTCGTAGTC

TTGGCGTTAT

CAAGACATTT

GCTGTCCCAG

CCATACCTGC

GTATTTTGTA

CATTAATGGA

GTCAATGTGA

GACAATGTGG

GGGCTGGATG

AACAGCCAGA

TTTATCATCT

CATTGACAAC

TTATGACAGA

CATCATTGTG

AGTCCTTCCG

TTTGAAGGAA

CATTCTGAAT

TTCTGGGAGT

ACAGACTCAG

AAGTGGCAAT

GAAATGCTTA

GATATTATAT

GTTTGAGAGC

TTGGCTCATT

TTCAACCAAC

AGAACAGAAG

ATTGTCTCTG

GACTCTACGA

TGAAGGTGGT

GTTTTGCTTG

ATACTTCTTT

TTATAAATTA

TTCTCTTGGA

CCTCGCTCTG

ATGCAGCTGT

AATTCACTCG

CTTCACTCTG

AGCAGAAAAA

AAATACTATA

801 851 AAAATTAGGA TCCAAAAAAC CTCAAAAACC

CATTCCACGG

0000 0* OS e 0 09 0 OS

S

0 0005** S S SOS 0 5 S OS S ft.tt, INFORMATION FOR SEQ lID NO:4: SEQUENCE CHARACTERISTICS: LENGTH: 702 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: RT-PCR DESCRIPTION: /desc =,,DNA probe/domain IV" (iii) HYPOTHETICAL:

NO

(iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: rat TISSUE TYPE: dorsal root ganglia CELL TYPE: peripheral nerve (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: 1 51 101 151 201 251 301 351 401 451 501 551 601 651 701 CTCAACATGG TTACGATGAT

GGTGGAGACC

GACGAAGGTT CTGGGCAGAA

TCAACCAGTT

GCGAGTGTGT GATGAAGATG

TTCGCCCTGC

GGCTGGAACG TGTTCGACTT

CATAGTGGTG

GCTGTTTCT GCAATCCTTA

AGTCACTGGA

TCTTCCGGGT CATCCGTCTG

GCCAGGATCG

CGAGCAGCCA AGGGGATTCG

CACGCTGCTC

GCCCGCCCTC TTCAACATCG

GCCTCCTCCT

ACTCCATCTT CGGCATGGCC

AGCTTCGCTA

ATCGACGACA TGTTCAACTT

CAAGACCTTT

GTTCCAGATC ACCACCTCGG

CCGGCTGGGA

TCAACACGGG GCCTCCCTAC

TGCGACCCCA

TCCCGGGGGA ACTGCGGGAG

CCCGGCGGTG

CTACATCATC ATCTCCTTCC

TCATCGTGGT

TC

GACGAGCAGG

CTTTGTGGCC

GACAGTACTA

ATCCTGTCCA

AAACTACTTC

GCCGCATCCT

TTCGCCCTCA

CTTCCTCGTC

ACGTCGTGGA

GGCAACAGCA

CGGCCTCCTC

ACCTGCCCAA

GGCATCATCT

CAACATGTAT

GCGAGGAGAA

GTCTTCACGG

TTTCACCAAC

TTGGGAGTCT

TCCCCGACGC

CAGGCTGATC

TGATGTCCCT

ATGTTCATCT

CGAGGCCGGC

TGCTGTGCCT

AGCCCCATCC

CAGCAACGGC

TCTTCACCAC

ATCGCAGTCA

0 0 #tOqO V INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 5334 base pairs TYPE: nucleic acid STRANDEDNESS: single 46 TOPOLOGY: linear (ii) MOLECULE TYPE: RT-PCR DESCRIPTION: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE:

ORGANISM:

TISSUE TYPE: CELL TYPE: (xi) SEQUENCE DESCRIPTION: SEQ ID 1 GTCGACTCTA

GATCAGGGTG

1 TTCCCGGACG AGCGGAATTT 101 151 201 251 301 351 401 451 501 551 601 651 701 751 801 851 901 951

CATAGAGAAG

AGGCGGCAGC

AGGAAGTTAC

GCCTCTGGAA

TGTTGAACAA

TTCATTCTGG

TGTCCATTCA

GTATGTTCAT

GAATACGTCT

GGCAAGAGGC

ACTGGCTGGA

GGCAGCCAAG

TCTGAAGGCG

TGCTGCGCTC

TGCCTCAGCA

GAACCAGAAG

AGGATTGCTT

ACCTGGCTCG

CGGATTGCTA

TGAGCCCCAG

CTAAGCTTTA

GACCTGGACC

GAAGAGAACA

GGCCTTTTAA

GTCTTTAGCA

GGCGAATTCT

TCATTGGGAT

TTCATTGTGG

CTTCATTGTC

TCAATCTTTC

ATTTCAGTTA

GGTGAAGAAG

TCTTTGCCCT

TGTATTAAGC

TGAAAAGGAA

GCAGCAGACC

AAGATGGAGG AGAGGTACTA

CCCGGTGATC

CCGCCCCTTC ACTTCCGACT CTCTGGCTGC TCCAAAAGGA GAGGAAGAAG

TCCAAAGACA

CCTCGGCCTC AGCTTGACCT AAAGGCCTCC TGGTGACATT CCCCCTGAGC TTGTAGCGAA CATTCTACAA AGACCATAAG

ACATTCATGG

ATTTATCGCT TCAGCGCCAA GCGGGCCTTG TCCCCTCAGA AGCTTAATGA

TTCGTATCTC

TGTTCATCAT CTGCACGGTG ATCATCAACT ATGGAGAGAA GTTTCGACAA CGACATTCCC TTATATTTTA GAAGCTGTGA

TTAAAATATT

ATGAGTTTTC CTTCCTCCGA

GATCCCTGGA

ATTGGAACAG CGATCGCAAC TTGTTTTCCG AGCTCTTCGT ACCTTCCGAG TGTTCAGAGC TCTCAGGTCT GAAGGTCATC GTAGGTGCCC CTGGTAGACG TGATGGTCCT CACTCTCTTC GGTCGGTCAG CAGCTGTTCA TGGGAATTCT ACAACTGTGG CCCCAACCCT GCATCCAACA AAAGATAGCG AAGACTTCAT AATGTGTGGT CTGTCCCAAT GGTTCTACGT GCGATAAAAC 7 4* *9 S. 5 500800

S.

S

0g@e S 5 0 eS S V. V 1001 1051 1101 1151 1201 1251 1301 1351 1401 1451 1501 1551 1601 1651 1701 1751 1801 1851 1901 1951 2001 2051 2101 2151 2201 2251 2301

CACATTGAAC

CCTTTCTCGC

TACCGACAGA

GGTGGTCATC

TTGTCACCAT

GAGGCCAAGG

GAAGGAGGCT

TTCAAGCTTC

ACAAGAAAGT

GTCTGATTCA

CCAAACGACT

GACCCCCTCC

CACCATGCAG

AAAATTTGGC

TGCATAAAGA

GGCCATCACC

ACCACAACAT

GTTTTCACGG

CGACCCTTAC

TGGCCCTCCT

AATAGGTCTT

CAAATCCTGG

TGGGCGCGCT

TTTTCTGTGG

CTACGCCACC

ACCACTCCTT

AACATGTGGG

CCAGACAATA

CATGTTCCGG

TCCTGCGGAC

TTCCTGGGCT

GGCTTATGAA

AGAAAATGTT

CTGGTTGCCA

ATCCTTTTCC

CCTTCTTTAT

GAGGACGATG

GTCCCAGAAC

ACAGGCAGAG

GAACAAGAAA

CTCTAAGTAC

AGGTCCTGCG

ATCTGCATCA

GGATGACAAC

GAATTTTCAT

CACTACTTCC

GAGTCTCGCT

TCTTGGCTTC

CCCACGTTAA

TGGAAACCTG

TGGGCATGCG

CAGGAGCGGC

CCTGGTGGTG

GCTGCATGCA

ATTATACAAA

GTTATGACTC

CTCTGGGATC

CCTTCTACCT

GAACAGAACA

TCAGGAAGCC

TGGGAATTGA

CCGAAGAAGA

GAGAGGGTCC

CCTCTAAAAA

TTGCCAGTGG

AGCGCTGAGC

AATTCCAGGA

CTGGTGTGGG

GACCATCATG

TCATCAATAC

TTAAAGACCA

AGCGGAAATG

GGCACGGCTG

GATGTGCTCT

CCTCAGAGTG

ACACTCTCAT

ACTGTGGTCC

GCTCTTCGGC

CCAGGCGGCG

TTCCGCATCC

GGATATGGAC

GTTTGACAAC

AAGACTCCTG

TACTTTGTCT

GCTTAACCTA

GAAATGTAGC

CAGCAGCTGT

CAGAAGTTCC

GGAAGTTTTT

AAGACGGCCC

TCCACAGCTC

ATCTCTTTGA

GCTGTCAGTA

GCCTTGTTTC

ACTGTAGCCC

ACGGATCCCT

CGTTTTCTTA

TACTGAAAAT

TGTCTCAAGA

GAATGTTTTT

ACAACACACT

CTGAGGGTCT

TAAGATCATC

TGACTATCGT

ACCAAGTTTA

CTGGCACATG

TCTGTGGGGA

GGCTCCCCGT

TTTGGCTGGT

GGAGAGGCTT

TCTTCTTCGT

ACCCTGGCTG

TGCTGAGACA

TAAGGGAGGA

CTTAATTCCC

CGGTAGTAAG

AAGCCTCAGC

CTTGAGCAGA

TGAGCACGTG

TCTTAACCAT

CCATGTGGGA

TCAGTGGCTG

TTACTGAGCT

GCCGTGGAGC

AGGAAACTGG

TCATCGCGCT

GACAGCATCG

GTCTGATAAC

TCAAGTTAGC

GGCCACTCCG

GGTCTTCATC

ACAAGACCGC

GATAATTTCT

ATGGATCGAG

TGTGCATCAT

2351 TGTCTTTGTC CTGATAATGG TGATCGGGAA GCTTGTGGTG

CTTAACCTCT

2401 2451 2501 2551 2601 2651 2701 2751 2801 2851 2901 2951 3001 3051 3101 3151 3201 3251 3301 2251 3401 3451 3501 3551 3601 3651 3701

TCATTGCCTU

CTGGAAGGAC

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

GCTGCTCAAT

TCCTTCAGCZ

AGACCAGGAA AACCAA.AGTC.

4 46 ,00, o

TTCTCCTTCP

GAGGAAAAAC

AGAATAAAGA

ACAGACATGG

CGCAGAGGTA

CCACCTCACA

ACCAAGCAGC

TTCTGAAGAA

ACGCAGTGAG

TTTAGAAATT

CTTTCCCAAG

GAAAGTCCCC

ATCGTGAAGC

GAGCAGTGGA

AAGTTGAGAA

CTCCTGGAAA

CACCAGTGCC

TCAGTCTCAT

CTGAGACCTC

CTACGCCCTG

GCCTCATTTT

GGGAAGTTTG

TTTTACCGAA

GGAAGGTCCC

CTGCTGCAAG

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

ATGAGGAGAJ

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

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

3751 TGAATGCTGC TGTCGATTCC

AGAGAGAAAG

49 ACGAGCAGCC GGACTTTGAG 3801 3851 3901 3951 4001 4051 4101 4151 4201 4251 4301 4351 4401 4451 4501 4551 4601 4651 4701 4751 4801 4851 4901 4951 5001 5051 5101

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

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

CTACTTTGTG

TTATCGGTGT

GGCCAAGACA

GAAAAAGTTA

ACAA.ATGTCA

GTCATCATTC

ATCTGCCGAC

TAGCCTTCGT

TTGAGGCAAC

CGTGGTTCTT

ACATTTCTTT

GGTCGAATCC

CTTTGCTTTG

TCTTCCTGGT

AAAGTGAAGA

TACGGGCAGC

ATACCCTCCT

TCCCAAGACA

TTACATCATC

TCCTCGAGAA

GAGGACGACT

GGCGTCGCAG

TGCCGGAGCC

ATGGACTTGC

CTTTGCTTTC

TGAAAACCAT

TCTACGAGC(

GTTTTTATCA

TATTATTGAC

TCTTCATGAC

GGAACCAAGA

AGCCTTTGTG

TGGGTCTTAT

CAGCCCAAAG

GGTCATCTTT

ACTACTTCAC

TCTATCATTA

CCCGCCCACG

TCAGGCTGGT

ATGATGTCTC

GATGTTCATT

AGGGCTCCGG

ATGCTGTGCC

CAACCCCATG

GCTGTCAGCA

ATCTCCTTCC

CTTCAACACA

TTGAAATCTT

TTCATCCAGT

GTTGCGTGTG

CCATGGTGAT

kCTACCAGGG 3ATGGAGGAG

CATAGTCAC

TCTTCGGCTC

AACTTCAATC

TGAGGAGCAG

AACCTCAAAA

TTCGACCTGG

TGTCTTAAAT

ATGTGAAGAA

ACCATAGAGT

CAATGGCTGG

GTACCCTGGT

CTCTTCAGAG

CCGGGCTGCC

TCCCCTCTCT

TACGCCATCT

GATCGACGAC

TCTTCCAGAT

CTGGAGGCAA

GCCGCAGATA

TCATCGTGGT

GCCACGGAGG

CTATGAGGTC

ATTCGGCCCT

GCCAAGCCGA

GGGCGACCGC

TCCTCGGGGA

AAGTTTATGG

CACCACCAAG

S

S

t *0*O It

I,

ee& a a 6* a a 5151 AGGAAGGAGG AGGAGCAAGG CGCCGCCGTC ATCCAGAGGG

CCTACCGGAA

5201 ACACATGGAG AAGATGGTCA AACTGAGGCT GAAGGACAGG

TCAAGTTCAT

5251 CGCACCAGGT GTTTTGCAAT GGAGACTTGT CCAGCTTGGA

TGTGGCCAAG

5301 GTCAAGGTTC ACAATGACTG AACCCTCATC

TAGA

Claims (17)

1. An isolated DNA sequence comprising the nucleotide sequence set forth in SEQ ID NO: 1 or SEQ ID NO:3.

2. The DNA of Claim 1 wherein said DNA sequence is encoding a sodium channel protein or fragment thereof.

3. The DNA of Claim 2 wherein said sodium channel protein is the oc-subunit or fragment thereof.

4. 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.

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.

8. The DNA of Claim 1 wherein said DNA is cDNA.

9. The DNA of Claim 1 wherein said DNA is synthetic DNA. Expression vectors comprising the DNA of Claim 8.

11. Expression vectors comprising the synthetic DNA of Claim 9.

12. Host cells transformed with the expression vectors of Claim

13. Host cells transformed with the expression vectors of Claim 11.

14. 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.

16. A tetrodotoxin-resistant sodium channel protein encoded by a DNA of Claims 1 to 9 or allelic variants thereof.

17. The protein of Claim 16 having the amino acid sequence set forth in SEQ ID NO:2.

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.

19. Poly- and/or monoclonal antibodies raised against a tetrodotoxin-resistant sodium channel •protein encoded by a DNA of Claims 1 to 9 or allelic variants thereof. A diagnostic kit comprising a polynucleotide of claim 14 capable of specifically S, hybridizing to a tetrodotoxin-resistant sodium channel protein or fragment thereof.

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. a 22. The invention substantially as hereinbefore described especially with reference to the foregoing Examples. Dated 17 November, 1998 F. Hoffman-La Roche AG Patent Attorneys for the Applicant/Nominated Person SPRUSON FERGUSON