CA2319143A1 - Modulation of sodium channels in dorsal root ganglia - Google Patents

Abstract

Description

Modulation of Sodium Channels in Dorsal Root Ganglia by Snlayman Dib-Hajj and Stephen G. Waxman FIELD OF THE INVENTION
The present invention relates to a novel tetrodotoxin resistant sodium 5 channel and related nucleotides, as well as screening assays for identifying agents useful in treating acute or chronic pain or other hyperexcitability states.
This application is related to U.S. Provisional Application 60/072,990, filed January 29, 1998, U.S. Provisional Application 60/109,402 entitled "Modulation of Sodium Channels in Dorsal Root Ganglia", filed November 20, 10 1998 and to U.S. Provision Application 60/109,666, entitled "Dii~erential Role of GDNF and NGF in the Maintenance of Two TTX-Resistant Sodium Channels in Adult DRG Neurons," filed on November 20, 1998, all of which are herein incorporated by reference.
BACKGROUND
15 A. 1~~]18~~
Voltage-gated sodium channels are a class of specialized protein molecules that act as molecular batteries permitting excitable cells (neurons and muscle fibers) to produce and propagate electrical impulses. Voltage-gated Na+
channels from rat brain are composed of three subunits, the pore-forming a 20 subunit (260 KDa) and two auxiliary subunits, X31 (36 KDa) and ~i2 (33 KDa) that may modulate the properties of the a-subunit; the a subunit is sufficient to form a functional channel that generates a Na current flow across the membrane [references 1,2 as cited below]. Nine distinct a subunits have been identified in vertebrates and are encoded by members of an expanding gene family [3 and 25 references therein, 4-6] and respective orthologues of a number of them have been cloned from various mammalian species including humans. Specific a subunits are expressed in a tissue- and developmentally-specific manner [7,8].

Aberrant expression patterns or mutations of voltage-gated sodium channel a-subunits underlie a number of human and animal disorders [9-13].
Voltage-gated sodium channel a-subunits consist of four domains (D1-4) of varying internal homology but of similar predicted structure, 5 connected by three intracellular loops (L1-3): The four domains fold to form a channel that opens to both the cytoplasm and the extracellular space via a pore.
The pore opens and closes depending upon the physiological state of the cell membrane.
Each domain consists of six transmembrane segments (Sl-6) that allow the protein to weave through the membrane with infra- and extracellular linkers.
The linkers of SS-S6 segments of the four domains contain sequences that line the pore of the channel, and a highly conserved subset of amino acids that acts as a filter to selectively allow sodium ions to traverse the channel pore into the cytoplasm, thus generating an electric current. The amphiphatic S4 segment, in each of the four domains, rich in basic residues repeated every third amino acid, acts as a voltage sensor and undergoes a conformational change as a result of the change in the voltage difference across the cell membrane. This in turn triggers the conformational change of the protein to open its pore to the extracellular Na+ ion gradient.
20 In most of the known voltage-gated sodium channel a-subunits, the channels close and change into an inoperable state quickly (inactivate), within a few milliseconds, after opening of the pore (activation); SNS-type channels, on the other hand, inactivate slowly and require a greater voltage change to activate. L3, the loop that links domains D3 and D4, contains a tripeptide which acts as an intracellular plug that closes the pore after activation, thus inducing the channel to enter the inactive state. After inactivation, these channels further undergo conformational change to restore their resting state and become available for activation. This period is referred to as recovery from inactivation (repriming). Different channels reprime at different rates, and repriming in SNS is relatively rapid.

wo ~r~sss9 rc~rms~rozoos Based on amino acid similarities, the voltage-gated sodium channel family has been further subdivided into two subfamilies [14J. Eight of the nine cloned channels belong to subfamily 1. They share many structural features, particularly in their S4 hansmembrane segments. However, some of them have 5 been shown to have distinct kinetic properties of inactivation and repriming.
Only a single channel of subfamily 2, also referred to as atypical channels, has been identified in human, rat and mouse tissues. This subfamily is primarily characterized by reduced numbers of basic residues in its S4 segments, and thus is predicted to have different voltage-dependence compared to subfamily 1. The 10 physiological function of subfamily 2 channels is currently unknown because its electrophysiological properties have not yet been elucidated.
The blocking of voltage-gated sodium channels by tetrodotoxin, a neurotoxin, has served to functionally classify these channels into sensitive (TTX-S) and resistant (TTX-R) phenotypes. Two mammalian TTX-R channels 15 have so far been identified, one specific to the cardiac muscle and to very limited areas of the central nervous system (CNS) and the second, SNS, is restricted to peripheral neurons (PNS) of the dorsal root ganglia (DRG) and trigeminal ganglia. Specific amino acid residues that confer resistance or sensitivity to TTX have been localized to the ion selectivity filter of the channel 20 pore. The SNS channel is also described in International Patent Application WO 97/01577.
B. Role of Sodium Channels in Disease States Because different Na+ channel a-subunit isotypes exhibit different kinetics and voltage-dependence, the firing properties of excitable cells depend 25 on the precise mixture of channel types that they express. Mutants of the cardiac and skeletal muscle a-subunit have been shown to cause a number of muscle disorders. Some examples are as follows: A change of a single basic amino acid residue in the S4 of the skeletal muscle channel is sufficient to change the kinetic properties of this channel and induce a disease state in many patients. A tripeptide deletion in L3 of the cardiac channel, proximal to the inactivation gate, induces a cardiac disorder called Long QT syndrome. A
single amino acid change in the SS-S6 linker of domain 1 of Scn8a, the region lining the pore of the channel, causes the mouse mutant "jolting". The total loss 5 of this channel by a different mutation causes motor end plate "med" disease in mice. This mutation is characterized by loss of motor neuron stimulation of the innervated muscle.
C. Sodium Channels and Pain Axonal injury (injury to nerve fibers, also called axons) can produce 10 chronic pain (termed neuropathic pain). A number of studies have demonstrated altered excitability of the neuronal cell body and dendrites after axonal injury [15-17], and there is evidence for a change in Na+ channel density over the neuronal cell body and dendrites following axonal injury [18-20]. The expression of abnormal mixtures of different types of sodium channels in a 15 neuronal cell can also lead to abnormal firing [13], and can contribute to hyperexcitability, paresthesia or pain.
Rent studies from our group on rat sensory DRG neurons have demonstrated a dramatic change in the expression profile of TTX-R and TTX-S
currents and in a number of mRNA transcripts that could encode the channels 20 responsible for these currents in DRG neurons following various insults [21-23].
We have, for example, shown an attenuation of the slowly inactivating, TTX-R
current and simultaneous enhancement of the rapidly inactivating, TTX-S Na+
currents in identified sensory cutaneous afferent neurons following axotomy [21]. We also have shown a loss of TTX-S, slowly repriming current and 25 TTX-R current and a gain in TTX-S, rapidly repriming current in nociceptive (pain) neurons following axotomy [22], down-regulation of SNS transcripts and a simultaneous up-regulation of a-III Transcripts [23]. Also associated with axotomy is a moderate elevation in the levels of aI and aII mRNAs [24].
These changes in the sodium channel profile appear to contribute to abnormal wo ~r~sss9 Pcrius99roioos firing that underlies neuropathic pain that patients suffer following axonal injury.
Inflammation, which is also associated with pain (termed inflammatory pain), also causes alteration in the sodium current profile in nociceptive DRG
5 neurons. Inflammatory modulators up-regulate TTX-R current in small C-type nociceptive DRG neurons in culture [25,26]. The rapid action of these modulators suggests that their action include posth~anslational modification of existing TTX-R channels. We have now determined that inflammation also increases a TTX-R Na+ current and up-regulates SNS transcripts in C-type DRG
10 neurons [58]. This data suggests that changes in the sodium current profile contribute to inflammation evoked-pain.
D. Therayies for Chronic Pain:
A variety of classes of drugs (anticonvulsants such as phenytoin and carbamazepine; anti-arrhythmics such as mexitine; local anesthetics such as 15 lidocaine) act on Na+ channels. Since the various Na+ channels produce sodium currents with different properties, selective blockade or activation (or other modulation) of specific channel subtypes is expected to be of significant therapeutic value. . Moreover, the selective expression of certain a-subunit isoforms (PNI, SNS; NaN) in specific types of neurons provides a means for 20 selectively altering their behavior.
Nociceptive neurons of the DRG are the major source of the PNS
TTX-R Na+ current. Thus, the Na+ channels producing TTX-R currents pmvide a relatively specific target for the manipulation of pain producing neumns. The molecular structure of one TTX-R channel in these DRG
25 neurons, SNS, has been identified but, prior to our research, it has not been determined whether there are other TTX-R channels in these neurons. If such channels could be identified, they would be ideal candidates as target molecules that are preferentially expressed in nociceptive neurons, and whose modulation would attenuate pain transmission.

SUMMARY OF THE INVENTION
The present invention includes an isolated nucleic acid which encodes a voltage gated Na+ channel that is preferentially expressed in dorsal root ganglia or trigeminal ganglia (the NaN channel). (In our preceding U.S. Provisional Application 60/072,990, this NaN channel was referred to by its previous name "NaX.") In a preferred embodiment, the isolated nucleic acid comprises the sequence shown in Fig. 1, Fig. 7A, Fig. 8A, allelic variants of said sequences or nucleic acids that hybridize to the foregoing sequences under stringent conditions.
In another embodiment, the invention includes an expression vector comprising an isolated nucleic acid which encodes the voltage gated Na+
channel that is preferentially expressed in dorsal root ganglia or trigeminal ganglia either alone or with appropriate regulatory and expression control elements. In a preferred embodiment, the expression vector comprises an 15 isolated nucleic acid having the sequence shown in Fig. 1, Fig. 7A, Fig.
8A, allelic variants of said sequences or nucleic acids that hybridize to the foregoing sequences under stringent conditions.
The present invention further includes a host cell transformed with an expression vector comprising an isolated nucleic acid which encodes a voltage gated Na+ channel that is preferentially expressed in dorsal root ganglia or trigeminal ganglia with appropriate regulatory and expression control elements.
In a preferred embodiment, the expression vector comprises an isolated nucleic acid having the sequence shown in Fig. 1, Fig. 7A, Fig. 8A, allelic variants of said sequences or nucleic acids that hybridize to the foregoing sequences under stringent conditions.
The present invention also includes an isolated voltage gated Na+
channel that is preferentially expressed in dorsal root ganglia or trigeminal ganglia. In a preferred embodiment, the channel has the amino acid sequence of Figs. 2, 7B or 8B or is encoded by a nucleic acid having the sequence shown in Figs. 1, 7A or 8A, allelic variants of said sequences or nucleic acids that WO 99!38889 PCT/US99/02008 hybridize to the foregoing sequences under stringent conditions. Peptide fragments of the channel are also included.
Another aspect of the invention is a method to identify an agent that modulates the activity of the NaN channel, comprising the steps of bringing the agent into contact with a cell that expresses the Na+ channel on its surface and measuring depolarization, or any resultant changes in the sodium current. The measuring step may be accomplished with voltage clamp measurements, by measuring depolarization, the level of intracellular sodium or by measuring sodium influx.
Another aspect of the invention is a method to identify an agent that modulates the transcription or translation of mRNA encoding the NaN channel.
The method comprises the steps of bringing the agent into contact with a cell that expresses the Na+ channel on its surface and measuring the resultant level of expression of the Na+ channel.
The invention also includes a method to treat pain, paraesthesia and hyperexcitability phenomena in an animal or human subject by administering an effective amount of an agent capable of modulating, such as by inhibiting or enhancing, Na+ current flow through NaN channels in DRG or trigeminal neurons. The method may include administering an effective amount of an 20 agent capable of modulating the transcription or translation of mRNA
encoding the NaN channel.
Another aspect of the invention is an isolated nucleic acid that is antisense to the nucleic acids described above. In a preferred embodiment, the antisense nucleic acids are of sufficient length to modulate the expression of NaN channel mRNA in a cell containing the mRNA.
Another aspect of the invention is a scintigraphic method to image the loci of pain generation or provide a measure the level of pain associated with DRG or trigeminal neuron mediated hyperexcitability in an animal or human subject by administering labeled monoclonal antibodies or other labeled ligands specific for the NaN Na+ channel.

_g_ Another aspect of the invention is a method to identify tissues, cells and cell types that express the NaN sodium channel. This method comprises the step of detecting NaN on the cell surface, or en route to the cell surface, or the presence of NaN encoding mRNA.
5 The present invention further includes a method of producing a transformed cell that expresses an exogenous NaN encoding nucleic acid, comprising the step of transforming the cell with an expression vector comprising an isolated nucleic acid having the sequence shown in Figs. 1, 7A
or 8A, allelic variants of said sequences or nucleic acids that hybridize to the 10 foregoing sequences under stringent conditions, together with appropriate regulatory and expression control elements. The invention also includes a method of producing recombinant NaN protein, comprising the step of culturing the transfornned host under conditions in which the NaN sodium channel or protein is expressed, and recovering the NaN protein.
15 The invention also includes an isolated antibody specific for the NaN
channel or polypeptide fragment thereof. The isolated antibody may be labeled.
Another aspect of the invention includes a therapeutic composition comprising an effective amount of an agent capable of decreasing rapidly repriming sodium current flow in axotomized, inflamed or otherwise injured 20 DRG neurons or in normal DRG neurons that are being driven to fire at high frequency. The invention also includes a method to treat acute pain or acute or chronic neuropathic or inflammatory pain and hyperexcitability phenomena in an animal or a human patient by administering the therapeutic composition.
The present invention also includes a method to screen candidate 25 compounds for use in treating pain and hyperexcitability phenomena by testing their ability to alter the expression or activity of an NaN channel mRNA or protein in axotomized, inflamed or otherwise injured DRG neurons.
BRIEF DESCRIPTION OF THE DRAWING FIGURES
Fig. 1 shows the sequence of the rat NaN cDNA.

Fig. 2 shows the putative amino acid sequence of the rat NaN cDNA.
Predicted transmembrance segments of domains I - IV are underlined. The amino acid serine "S" in DI-SS2, implicated in the TTX-R phenotype, is in bold face type.
5 Fig. 3 presents a schematic diagram of predicted secondary structure of the NaN a-subunit.
Fig. 4 shows the results of RT-PCR analysis for a-NaN in extracts of various tissues using NaN specific primers. NaN is abundantly expressed in dorsal root and trigeminal ganglia. Low levels of NaN are detected in cerebral hemisphere 10 and retina tissues. No detectable NaN signal is seen in cerebellum, optic nerve, spinal cord, sciatic nerve, superior cervical ganglia, skeletal muscle, cardiac muscle, adrenal gland, uterus, liver and kidney.
Fig. 5 shows the tissue distribution of a-NaNby in situ hybridization. A.
Trigeminal ganglion neurons show moderate-to-high hybridization signal. B.
15 Dorsal root ganglion neurons show moderate-to-high hybridization signal in small neurons. Hybridization signal is attenuated in large neurons (arrow). C.
Sense probe shows no signal in DRG neurons. D., E., and F. No hybridization signal is seen in spinal cord, cerebellum and liver. All tissues are from adult Sprague-Dawley rat. Scale bars = 50 micrometer.
20 Fig. 6 shows the predicted lengths of domain I amplification products of rat a-subunits and their subunit-specific restriction enzyme profile.
Figs. 7A-7B set forth the nucleotide and amino acid sequences of the marine NaN.
Fig. 8A-Fig.BB. Fig.BA is a partial nucleotide sequence of the human NaN.
25 Fig. 8B is a partial amino acid sequence of the human NaN protein.
Fig. 9 shows cultures of DRG neurons obtained from L4/5 ganglia of adult rats that were reacted with antibody to NaN and then processed for immunofluorescent localization. a.,b. NaN immunostaining is prominent within the cell bodies of DRG neurons. c. NaN is present in the neuritic outgrowths, as -lU-well as the cell bodies, of DRG neurons. d., d'. Nomarski (d.) and fluorescent (d'.) images of a neuron that does not express NaN protein.
Fig. 10 shows the location of Scnl la and related genes on distal mouse chromosome 9. (A) Haplotypes from the Jackson BSS backcross. Black boxes 5 represent C57BL/6J alleles and white boxes represent SPRET/Ei alleles. The number of animals with each haplotype is given below each column. Missing data was inferred finm adjacent data when typing was ambiguous. (B) Map of distal chromosome 9 based on data in (A). Positions of ScnSa and ScnlOa from the MGD consensus map and the locations of the human orthologs are 10 indicated. Numbers are cM positions on the consensus map (http://www.informatics jax.org/bin/ccr/index).
DETAILED DESCRIPTION
The present invention relates to a novel gene that we have discovered, called NaN. NaN encodes a previously unidentified protein, referred to herein 15 as NaN, that belongs to the a-subunit voltage-gated sodium channel protein family and that produces a TTX-R sodium current. Such channels underlie the generation and propagation of impulses in excitable cells like neurons and muscle fibers. NaN is a novel sodium channel, with a sequence distinct finm other, previously identified, channels. The preferential expression of NaN on 20 sensory, but not other neurons, makes it a very useful target for diagnostic and/or therapeutic uses in relation to acute and/or chronic pain pathologies.
Definitions:
This specification uses several technical terms and phrases which are intended to have the following meanings:
25 The phrase "modulate" or "alter" refers to up- or down-regulating the level or activity of a particular receptor, ligand or current flow. For example an agent might modulate Na+ current flow by inhibiting (decreasing) or enhancing (increasing) Na+ current flow. Similarly, an agent might modulate the level of WO 99!38889 PCT/US99/02008 expression of the NaN sodium channel or the activity of the NaN channels that are expressed.
The phrase "sodium current" or "Na+ current" means the flow of sodium ions across a cell membrane, often through channels (specialized pmtein S molecules) that are specifically permeable to certain ions, in this case sodium ions.
The phrase "voltage gated" means that the ion channel opens when the cell membrane is in a particular voltage range. Voltage-sensitive sodium channels open when the membrane is depolarized. They then permit Na* ions to 10 flow into the cell, producing further depolarization. This permits the cell to generate electrical impulses (also known as "action potentials'.
The phrase "rapidly repriming" means that the currents recover from inactivation more rapidly than do such currents in most other voltage gated sodium channel family members.
15 The terms "TTX-R" and '"TTX-S" means that the flow of current through a cell membrane is, respectively, resistant or sensitive to tetrodotoxin (a neurotoxin produced in certain species) at a concentration of about I00 nM.
The phrase "peripheral nervous system (PNS)" means the part of the nervous system outside of the brain and spinal cord, i.e., the spinal roots and 20 associated ganglia such as dorsal root ganglia (DRG) and trigeminal ganglia, and the peripheral nerves.
The phrase "inhibits Na+ current flow" means that an agent has decreased such current flow relative to a control cell not exposed to that agent.
A preferred inhibitor will selectively inhibit such current flow, without affecting 25 the current flow of other sodium channels; or it will inhibit Na+ current in the channel of interest to a much larger extent than in other channels.
The phrase "enhances Na* current flow" means that an agent has increased such current flow relative to a control cell not exposed to that agent.
A preferred agent will selectively increase such current flow, without affecting WO 99/38889 PC1'/US99/0?.008 the current flow of other sodium channels; or it will increase Na+ current in the channel of interest to a much larger extent than in other channels.
The phrase "specifically hybridizes" refers to nucleic acids which hybridize under highly stringent or moderately stringent conditions to the 5 nucleic acids encoding the NaN sodium channel, such as the DNA sequence of Figs. 1, 7A or 8A.
The phrase "isolated nucleic acid" refers to nucleic acids that have been separated from or substantially purified relative to contaminant nucleic acids encoding other polypeptides. "Nucleic acids" refers to all fortes of DNA and 10 RNA, including cDNA molecules and antisense RNA molecules.
The phrase "RT-PCR" refers to the process of reverse transcription of RNA (RT) using the enzyme reverse transcriptase, followed by the amplification of certain cDNA templates using the polymerise chain reaction (PCR); PCR requires generic or gene-specific primers and thermostable DNA
15 polymerise, for example, Taq DNA polymerise.
The phrase "preferentially expressed" means that voltage gated Na+
channel is expressed in the defined tissues in detestably greater quantities than in other tissues. For instance, a voltage gated Na+ channel that is preferentially expressed in dorsal root ganglia or trigeminal ganglia is found in detestably 20 greater quantities in dorsal root ganglia or trigeminal ganglia when compared to other tissues or cell types. The quantity of the voltage gated Na+ channel may be detected by any available means, including the detection of specific RNA
levels and detection of the channel protein with specific antibodies.
25 The present invention relates to a previously unidentified, voltage-gated sodium channel a-subunit (NaN), predicted to be TTX-R, voltage-gated, and preferentially expressed in sensory neurons innervating the body (dorsal root ganglia or DRG) and the face (trigeminal ganglia). The predicted open reading frame (ORF), the part of the sequence coding for the NaN protein molecule, has been determined with the putative amino acid sequence from different species (rat, mouse, human) presented in Figs. 2, 7B and 8B.
All of the relevant landmark sequences of voltage-gated sodium channels are present in NaN at the predicted positions, indicating that NaN
belongs to the sodium channel family. But NaN is distinct from all other previously identified Na channels, sharing a sequence identity of less than 53%
with each one of them. NaN is distinct from SNS, the anly other TTX-R Na+
channel subunit that has been identified, until our discovery, in PNS. We have identified and cloned NaN without using any primers or probes that are based 10 upon or specific to SNS. Moreover, NaN and SNS share only 47% similarity of their predicted open reading fi~ame (ORF), comparable to the limited similarity of NaN to all subfamily 1 members.
The low sequence similarity to existing a-subunits clearly identifies NaN as a novel gene, not simply a variant of an existing channel. Sequence 15 variations compared to the other voltage-gated channels indicate that NaN
may be the prototype of a novel and previously unidentified, third class of TTX-R
channels that may possess distinct properties compared to SNS. NaN and SNS, which are present in nociceptive DRG and trigeminal neurons, may respond to pharmacological interventions in different ways. The preferential expression of 20 NaN in sensory DRG and trigeminal neurons provides a target for selectively modifying the behavior of these nerve cells while not affecting other nerve cells in the brain and spinal cord. A further elucidation of the properties of NaN
channels will be important to understand more fully the effects of drugs designed to modulate the function of the "TTX-R" currents which are 25 characteristic of DRG nociceptive neurons and which contribute to the transmission of pain messages, and to abnormal firing patterns after nerve injury and in other painful conditions.

Nucleic acid molecules of the invention include the nucleotide sequences set forth in Fig. 1, Fig. 7A, Fig. 8A as well as nucleotide sequences that encode the amino acid sequences of Fig. 2, Fig. 7B and Fig. 8B. Nucleic acids of the claimed invention also include nucleic acids which specifically hybridize to nucleic acids comprising the nucleotide sequences set forth in Fig.
1, Fig. 7A or 8A or nucleotide sequences which encode the amino acid sequences of Fig. 2, Fig. 7B or Fig.BB. A nucleic acid which specifically hybridizes to a nucleic acid comprising that sequence remains stably bound to said nucleic acid under highly stringent or moderately stringent conditions.
Stringent and moderately stringent conditions are those commonly defined and available, such as those defined by Sambrook et al. [59] or Ausubel et al.
[60].
The precise level of stringency is not important, rather, conditions should be selected that provide a clear, detectable signal when specific hybridization has occurred.
Hybridization is a function of sequence identity (homology), G+C
content of the sequence, buffer salt content, sequence length and duplex melt temperature (T[m]) among other variables. See, Maniatis et al.[62]. With similar sequence lengths, the buffer salt concentration and temperature provide useful variables for assessing sequence identity (homology) by hybridization techniques. For example, where there is at least 90 percent homology, hybridization is commonly carried out at 68° C in a buffer salt such as diluted from 20XSSC. See Sambrook et al. [59]. The buffer salt utilized for final Southern blot washes can be used at a low concentration, e.g., O.1XSSC
and at a relatively high temperature, e.g., 68° C, and two sequences will form a 25 hybrid duplex (hybridize). Use of the above hybridization and washing conditions together are defined as conditions of high stringency or highly stringent conditions. Moderately stringent conditions can be utilized for hybridization where two sequences share at least about 80 percent homology.
Here, hybridization is carried out using 6XSSC at a temperature of about 50-55° C. A final wash salt concentration of about 1-3XSSC and at a temperature of about 60-68° C are used. These hybridization and washing conditions define moderately stringent conditions.
In particular, specific hybridization occurs under conditions in which a high degree of complementarity exists between a nucleic acid comprising the 5 sequence of an isolated sequence and another nucleic acid. With specific hybridization, complementarity will generally be at least about 70%, 75%, 80%, 85%, preferably about 90-100%, or most preferably about 95-100%.
As used herein, homology or identity is determined by BLAST (Basic Local Alignment Search Tool) analysis using the algorithm employed by the 10 programs blastp, blasts, blasts, tblastn and tblasta (Karlin et al. Proc.
Natl.
Acad. Sci. USA 87: 2264-2268 (1990) and Altschul, S. F. J. Mol. Evol. 36:
290-300(1993), both of which are herein incorporated by reference) which are tailored for sequence similarity searching. The approach used by the BLAST
program is to first consider similar segments between a query sequence and a 15 database sequence, then to evaluate the statistical significance of all matches that are identified and finally to summarize only those matches which satisfy a preselected threshold of significance. For a discussion of basic issues in similarity searching of sequence databases, see Altschul et al. (Nature Genetics 6: 119-129 (1994)) which is herein incorporated by reference. The search 20 parameters for histogram, descriptions, alignments, expect (i.e., the statistical significance threshold for reporting matches against database sequences), cutoff, matrix and filter are at the default settings. The default scoring matrix used by blastp, blastz, tblastn, and tblastx is the BLOSUM62 matrix (Henikoff et al. Proc. Natl. Acad. Sci. USA 89: 10915-10919 (1992), herein 25 incorporated by reference). For blasts, the scoring matrix is set by the ratios of M (i.e., the reward score for a pair of matching residues) to N (i.e., the penalty score for mismatching residues), wherein the default values for M and N are 5 and -4, respectively.
The nucleic acids of the present invention can be used in a variety of 30 ways in accordance with the present invention. For example, they can be used as nucleic acid probes to screen other cDNA and genomic DNA libraries so as to select by hybridization other DNA sequences that encode homologous NaN
sequences. Contemplated nucleic acid probes could be RNA or DNA labeled with radioactive nucleotides or by non-radioactive methods (for example, S biotin). Screening may be done at various stringencies (through manipulation of the hybridization Tm, usually using a combination of ionic strength, temperature and/or presence of formamide) to isolate close or distantly related homologs. The nucleic acids may also be used to generate primers to amplify cDNA or genomic DNA using polymerase chain reaction (PCR) techniques.
10 The nucleic acid sequences of the present invention can also be used to identify adjacent sequences in the genome, for example, flanking sequences and regulatory elements of NaN. The nucleic acids may also be used to generate antisense primers or constructs that could be used to modulate the level of gene expression of NaN. The amino acid sequence may be used to design and 15 produce antibodies specific to NaN that could be used to localize NaN to specific cells and to modulate the function of NaN channels expressed on the surface of cells.
Vectors and Transform~e~~ Host Cells:
The present invention also comprises recombinant vectors containing and 20 capable of replicating and directing the expression of nucleic acids encoding a NaN sodium channel in a compatible host cell. For example, the insertion of a DNA in accordance with the present invention into a vector using enzymes such as T4 DNA ligase, may be performed by any conventional means. Such an insertion is easily accomplished when both the DNA and the desired vector 25 have been cut with the same restriction enzyme or enzymes, since complementary DNA termini are thereby produced. If this cannot be accomplished, it may be necessary to modify the cut ends that are produced by digesting back single-stranded DNA to produce blunt ends, or by achieving the same result by filling in the single-stranded termini with an appropriate DNA

polymerase. In this way, blunt-end ligation may be carried out. Alternatively, any site desired may be produced by ligating nucleotide sequences (linkers) onto the DNA termini. Such linkers may comprise specific oligonucleotide sequences that encode restriction site recognition sequences.
5 Any available vectors and the appropriate compatible host cells may be used [59, 60]. Commercially available vectors, for instance, those available from New England Biolabs Inc., Promega Corp., Stratagene Inc. or other commercial sources are included.
The transformation of appropriate cell hosts with an rDNA (recombinant 10 DNA) molecule of the present invention is accomplished by well known methods that typically depend on the type of vector used and host system employed. Frog oocytes can be injected with RNA and will express channels, but in general, expression in a mammalian cell line (such as HEK293 or CHO
cells) is preferred. With regard to transformation of prokaryotic host cells, 15 electmporation and salt treatment methods are typically employed, see, for example, Cohen et al. [6l ]; and [62]. With regard to transformation of vertebrate cells with vectors containing rDNAs, electroporation, cationic lipid or salt treatment methods are typically employed [63, 64].
Successfully transformed cells, i.e., cells that contain an rDNA molecule 20 of the present invention, can be identified by well known techniques. For example, cells resulting from the introduction of an rDNA of the present invention can be cloned to produce single colonies. Cells from those colonies can be harvested, lysed and their DNA content examined for the presence of the rDNA using conventional methods [65, 66] or the proteins produced from the 25 cell assayed via an immunological method. If tags such as green fluorescent protein are employed in the construction of the recombinant DNA, the transfected cells may also be detected in vivo by the fluorescence of such molecules by cell sorking.
For transient expression of recombinant channels, transformed host cells 30 for the measurement of Na+ current or intracellular Na+ levels are typically wo ~r~8ss9 rc~rius~rozoos prepared by co-transfecting constructs into cells such as HEK293 cells with a fluorescent reporter plasmid (such as pGreen Lantern-1, Life Technologies, Inc.) using the calcium-phosphate precipitation technique [27]. HEK293 cells are typically grown in high glucose DMEM (Life Technologies, Inc) 5 supplemented with 10% fetal calf serum (Life Technologies, Inc). After 48 hrs, cells with green fluorescence are selected for recording [28].
For preparation of cell lines continuously expressing recombinant channels, the NaN construct is cloned into other vectors that carry a selectable marker in mammalian cells. Transfecdons are carried out using the calcium phosphate precipitation technique [27J. Human embryonic kidney (HEK-293), Chinese hamster ovary (CHO) cells, derivatives of either or other suitable cell lines are grown under standard tissue culture conditions in Dulbeccos's modified Eagle's medium supplemented with 10% fetal bovine serum. The calcium phosphate-DNA mixture is added to the cell culture medium and left for 15-20 hr, after which time the cells are washed with fresh medium. After 48 hrs, antibiotic (G418, Geneticin, Life Technologies) is added to select for cells which have acquired neomycin resistance. After 2-3 weeks in 6418, 10-20 isolated cell colonies are harvested using sterile l4ml pipette tips. Colonies are grown for another 4-7 days, split and subsequently tested for channel expression using whole-cell patch-clamp recording techniques and RT-PCR.
ethod ; f Measuring Na+ Current Flow:
Na+ currents are measured using patch clamp methods [29], as described by Rizzo et al. [30] and Dib-Hajj et al. [28J. For these recordings data are acquired on a Macintosh Quadra 950 or similar computer, using a program such as Pulse (v 7.52, HEKA, German). Fire polished electrodes typically (0.8-1.5 MVO are fabricated from capillary glass using a Sutter P-87 puller or a similar instrument. In the most rigorous analyses, cells are usually only considered for analysis if initial seal resistance is <5 Gohm, they have high leakage currents (holding current <0.1 nA at -80 m~, membrane blebs, and an access resistance WO 99!38889 PCTNS99/02008 <5 Mohm. Access resistance is usually monitored throughout the experiment and data are not used if resistance changes occur. Voltage errors are minimized using series resistance compensation and the capacitance artifact is canceled using computer controlled amplifier circuitry or other similar methods. For comparisons of the voltage dependence of activation and inactivation, cells with a maximum voltage ermr of tlOmV after compensation are used. Linear leak subtraction is usually used for voltage clamp recordings. Membrane currents are typically filtered at 5 KHz and sampled at 20 KHz. The pipette solution contains a standard solution such as: 140 mM CsF, 2 mM MgCI" 1 mM EGTA, 10 and 10 mM Na-HEPES (pH 7.3). The standard bathing solution is usually 140 nM NaCI, 3 mM KCI, 2 mM MgCl2, 1 mM CaCl2, 10 mM HEPES, and 10 mM
glucose (pH 7.3).
Voltage clamp studies on transformed cells or DRG neurons, using methods such as intracellular patch-clamp recordings, can provide a quantitative measure of the sodium current density (and thus the number of sodium channels in a cell), and channel physiological properties. These techniques, which measure the currents that flow through ion channels such as sodium channels, are described in Rizzo et al. [21J. Alternatively, the blockage or enhancement of sodium channel function can be measured using optical imaging with 20 sodium-sensitive dyes or with isotopically labeled Na. These methods which are described in Rose, et al., (J. Neurophysiology, 1997 in press) [67] and by Kimelberg and Walz [31 ), measure the increase in intracellular concentration of sodium ions that occurs when sodium channels are open.
h~easurement oflntracellular Sodium ~~Na*7.l 25 The effects of various agents on cells that express Na'" can be determined using ratiometric imaging of [Na+]i using SBFI or other sinular ion-sensitive dyes. In this method, as described by Sontheimer et al. [32], cytosolic-free Na+
is measured using an indicator for Na+, such as SBFI (sodium-binding benzofuran isophthalate; [33]) or a similar dye. Cells are first loaded with the membrane-permeable acetoxymethyl ester form of the dye (which is dissolved in dimethyl sulfoxide (DMSO) at a stock concentration of 10 mTV)7. Recordings are obtained on the stage of a microscope using a ratiometric imaging setup (e.g., finm Georgia Instruments). Excitation light is provided at appropriate 5 wavelengths (e.g., 340:385 nm). Excitation light is passed to the cells through a dichroic reflector (400 nm) and emitted light above 450 nm is collected.
Fluorescence signals are amplified, e.g., by an image intensifier (GenIISyS) and collected with a CCD camera, or similar device, interfaced to a frame grabber.
To account for fluorescence rundown, the fluorescence ratio 340:385 is used to 10 assay cytosolic-free Na*.
For calibration of SBFI's fluorescence, cells are perfused with calibration solutions containing known Na* concentrations (typically 0 and 30 mM, or 0, 30, and 50 mM [Na*]), and with ionophones such as gramicidin and monensin (see above) after each experiment. As reported by Rose and Ransom 15 [34], the 345/390 nm fluorescence ratio of intracellular SBFI changes monotonically with changes in [Na*];. Experiments are typically repeated on multiple (typically at least 4) different coverslips, providing statistically significant measurements of intracellular sodium in control cells, and in cells exposed to various concentrations of agents that may block, inhibit or enhance 20 Na*.
Method to Measure Na* Influx via Measuring; llNa or 86Rb.
~Na is a gamma emitter and can be used to measure Na* flux [31 ], and ~Rb*
can be used to measure Na*/K*-ATPase activity [32]. g6Rb* ions are taken up by the Na+/K*-ATPase-like K* ions, but have the advantage of a much longer 25 half life than ''2K* [35]. Thus, measurement of the unidirectional ouabain-sensitive ~Rb* uptake provides a quantitative method for assaying Na*/K*-ATPase activity which provides another indicator of the electrical firing of nerve cells. Following incubation of cells expressing NaN with the isotope ~Na*, the cellular content of the isotope is measured by liquid scintillation wo ~r~sss9 rcrius~rozoos counting or a similar method, and cell protein is determined using a method such as the bicinchoninic acid protein assay [36] following the modifications described by Goldschmidt and Kimelberg [37] for cultured cells. ~Na and g6Rb*
fluxes are determined in the presence and absence of agents that may block, S inhibit, or enhance NaN. This permits determination of the actions of these agents on NaN.
Method to Ident' '~A,gent<s that Modulate NaN Mediated Current:
Several approaches can be used to identify agents that are able to modulate (i.e., block or augment) the Na* current through the NaN sodium channel. In general, to identify such agents, a model cultured cell line that expresses the NaN sodium channel is utilized, and one or more conventional assays are used to measure Na* current. Such conventional assays include, for example, patch clamp methods, the ratiometric imaging of [Na*],, and the use of ~Na and ~Rb as described above.
In one embodiment of the present invention, to evaluate the activity of a candidate compound to modulate Na* current, an agent is brought into contact with a suitable transformed host cell that expresses NaN. After mixing or appropriate incubation time, the Na* current is measured to determine if the agent inhibited or enhanced Na* current flow.
20 Agents that inhibit or enhance Na* current are thereby identified. A
skilled artisan can readily employ a variety of art-recognized techniques for determining whether a particular agent modulates the Na* current flow.
Because Na* is preferentially expressed in pain-signaling cells, one can also design agents that block, inhibit, or enhance Na* channel function by measuring the response of laboratory animals, treated with these agents, to acute or chronic pain. In one embodiment of this aspect of the invention, laboratory animals such as rats are treated with an agent for instance, an agent that blocks or inhibits (or is thought to block or inhibit) NaN. The response to various painful stimuli are then measured using tests such as the tail-flick test and limb WO 99138889 PGTlUS99/02008 withdrawal reflex, and are compared to untreated controls. These methods are described in Chapter 1 S of Reference [38]. In another embodiment of this aspect of the invention, laboratory animals such as rats are subjected to localized injection of pain-producing inflammatory agents such as formalin 5 [39], Freunds adjuvant [40] or carageenan, or are subjected to nerve constriction [41,42] or nerve transection [43] which produce persistent pain. The response to various normal and painful stimuli are then measured, for example, by measuring the latency to withdrawal from a warm or hot stimulus [38] so as to compare control animals and animals treated with agents that are thought to modify NaN.
The preferred inhibitors and enhancers of NaN preferably will be selective for the NaN Na+ channel. They may be totally specific (like tetrodotoxin, TTX, which inhibits sodium channels but does not bind to or directly effect any other channels or receptors), or relatively specific (such as lidocaine which binds to and blocks several types of ion channels, but has a predilection for sodium channels). Total specificity is not required for an inhibitor or enhancer to be efficacious. The ratio of its effect on sodium channels vs. other channels and receptors, may often determine its effect and effects on several channels, in addition to the targeted one, may be efficacious [44].
It is contemplated that modulating agents of the present invention can be, as examples, peptides, small molecules, naturally occurring and other toxins and vitamin derivatives, as well as carbohydrates. A skilled artisan can readily recognize that there is no limit as to the structural nature of the modulating agents of the present invention. Screening of libraries of molecules may reveal agents that modulate NaN or current flow through it. Similarly, naturally occundng toxins (such as those produced by certain fish, amphibians and invertebrates) can be screened. Such agents can be routinely identified by exposing a transformed host cell or other cell which expresses a sodium channel to these agents and measuring any resultant changes in Na+ current Recombinant Protein Expression. Synthesis and Pur; cation:
Recombinant NaN pioteins can be expressed, for example, in E. coli strains HB 101, DHSa or the protease deficient strain such as CAG-4S6 and purified by conventional techniques.
S The peptide agents of the invention can be prepared using standard solid phase (or solution phase) peptide synthesis methods, as is known in the art.
In addition, the DNA encoding these peptides may be synthesized using commercially available oligonucleotide synthesis instrumentation and produced recombinantly using standard recombinant production systems. The production 10 using solid phase peptide synthesis is necessitated if non-gene-encoded amino acids are to be included.
Antibodies and Immunodetection:
Another class of agents of the present invention are antibodies immunoreactive with the Na+ channel. These antibodies may block, inhibit, or 1 S enhance the Na+ current flow through the channel. Antibodies can be obtained by immunization of suitable mammalian subjects with peptides, containing as antigenic regions, those portions of NaN, particularly (but not necessarily) those that are exposed extracellularly on the cell surface. Such immunological agents also can be used in competitive binding studies to identify second generation 20 inhibitory agents. The antibodies may also be useful in imaging studies, once appropriately labeled by conventional techniques.
Production of T~genic Animals:
Transgenic animals containing and mutant, knock-out or modified NaN
genes are also included in the invention. Transgenic animals wherein both NaN
2S and the SNS/PN3 gene are modified, disrupted or in some form modified are also included in the present invention. Transgenic animals are genetically modified animals into which recombinant, exogenous or cloned genetic material has been experimentally transferred. Such genetic material is often referred to as a "bransgene". The nucleic acid sequence of the transgene, in this case a form of NaN, may be integrated either at a locus of a genome where that particular nucleic acid sequence is not otherwise normally found or at the normal locus for the transgene. The transgene may consist of nucleic acid sequences derived 5 from the genome of the same species or of a different species than the species of the target animal.
The term "germ cell line transgenic animal" refers to a transgenic animal in which the genetic alteration or genetic information was introduced into a germ line cell, thereby confernng the ability of the transgenic animal to transfer 10 the genetic information to offspring. If such offspring in fact possess some or all of that alteration or genetic information, then they too are transgenic animals.
The alteration or genetic information may be foreign to the species of animal to which the recipient belongs, foreign only to the particular individual recipient, or may be genetic information already possessed by the recipient.
In 15 the last case, the altered or introduced gene may be expressed differently than the native gene.
Transgenic animals can be produced by a variety of different methods including tnansfection, electmporation, microinjection, gene targeting in embryonic stem cells and recombinant viral and retmviral infection (see, e.g., 20 U.S. Patent No. 4,736,866; U.S. Patent No. 5,602,307; Mullins et al. (1993) Hypertension 22(4):630-633; Brenin et al. (1997) Surg. Oncol. 6(2)99-110;
Tuan (ed.), Recombinant Gene Expression Protocols, Methods in Molecular Biology No. 62, Humans Press (1997)).
A number of recombinant or transgenic mice have been produced, 25 including those which express an activated oncogene sequence (U.S. Patent No.
4,736,866); express simian SV 40 T-antigen (U.S. Patent No. 5,728,915); lack the expression of interferon regulatory factor 1 (IItF-1) (U.S. Patent No.
5,731,490); exhibit dopaminergic dysfunction (U.S. Patent No. 5,723,719);
express at least one human gene which participates in blood pressure control 30 (U.S. Patent No. 5,731;489); display greater similarity to the conditions existing wo ~r~sss9 pcr~rs~rozoos in naturally occurring Alzheimer's disease (U.S. Patent No. 5,720,936); have a reduced capacity to mediate cellular adhesion (U.S. Patent No. 5,602,307);
possess a bovine growth hormone gene (Clutter et al. (1996) Genetics 143(4}:1753-1760); or, are capable of generating a fully human antibody response (McCarthy (1997) The Lancet 349(9049):405).
While mice and rats remain the animals of choice for most transgenic experimentation, in some instances it is preferable or even necessary to use alternative animal species. Transgenic procedures have been successfully utilized in a variety of non-marine animals, including sheep, goats, pigs, dogs, cats, monkeys, chimpanzees, hamsters, rabbits, cows and guinea pigs (see, e.g., Kim et al. (1997) Mol. Reprod. Dev. 46(4):515-526; Houdebine (1995) Reprod.
Nutr. Dev. 35(6):609-617; Petters (1994) Reprod. Fertil. Dev. 6(5):643-645;
Schnieke et al. (1997) Science 278(5346):2130-2133; and Ainoah (1997) J.
Animal Science 75(2):578-585}.
The method of introduction of nucleic acid fragments into recombination competent mammalian cells can be by any method which favors co-transformation of multiple nucleic acid molecules. Detailed procedures for producing transgenic animals are readily available to one skilled in the art, including the disclosures in U.S. Patent No. 5,489,743 and U.S. Patent No.
5,602,307.
The specific examples presented below are illustrative only and are not intended to limit the scope of the invention.
EXAMPLES
Example 1: Cloning and Characterization of the Rat NaN Codine a. RNA Preparation Dorsal root ganglia (DRG) from the lumber region (L4-LS) were dissected from adult Sprague-Dawley rats and total cellular RNA was isolated by the single step guanidinum isothiocyanate-acid phenol procedure [45]. For analytical applications, DRG tissues were dissected from a few animals at a time. The quality and relative yield of the RNA was assessed by elechophoresis in a 1% agarose gel. Because of the limited starting material (4 DRGs weigh on average 10 mg), quantifying the RNA yield was not attempted. PolyA+ RNA
was purified from about 300 pg of total DRG RNA (28 animals) using the PolyATract isolation system according to the manufacturers recommendations (Promega). Half of the purified RNA was used for the preparation of Marathon cDNA (see below) without further quantification.
b. Reverse Transcr~~tion For analytical applications, first strand cDNA was synthesized essentially as previously described [46]. Briefly, total RNA was reverse transcribed in a 25 ~1 final volume using 1 p,M random hexamer (Boehringer Mannheim) and 500 units Superscript II reverse transcriptase (Life Technologies) in the presence of 100 units of RNase Inhibitor (Boehringer Mannheim). The reaction buffer consisted of 50 mM Tris-HCl (pH 8.3), 75 mM KCI, 3 mM MgCl2, 10 mM DTT and 125 pM dNTP. The reaction was allowed to proceed at 37°C for 90 min., 42°C for 30 min, then terminated by heating to 65°C for 10 min.
c. First-Strand cDNA Synthesis The Marathon cDNA synthesis protocol was followed according to the manufacturer's instruction as summarized below (all buffers and enzymes are purchased from the manufacturer (Clontech):
Combine the following reagents in a sterile 0.5-ml microcentrifuge tube:
1 ~g (1-4 p.l) PolyA+ RNA sample, 1 p.l cDNA Synthesis Primer (10 ~.M) and sterile H20 to a final volume of 5 p.l. Mix contents and spin the tube briefly in a microcentrifuge. Incubate the mixture at 70°C for 2 min., then immediately quench the tube on ice for 2 min. Touch-spin the tube briefly to collect the wo ~r~sss9 pc~rius99rozoos condensation. Add the following to each reaction tube: 2 wl SX First-Strand Buffer, 1 pl dNTP Mix (10 mM), 1 ~1 [a-32PJdCTP (1 p,Ci/~1), 1 ~.l AMV
Reverse Transcriptase (20 units/~1) for a 10 ~1 volume. The radiolabeled dCTP
is optional (used to determine yield of cDNA) and is replaced by sterile HZO
if 5 not used. Mix the contents of the tube by gently pipetting and touch-spin the tube to collect the contents at the bottom. Incubate the mixture at 42°C for 1 hr in an air incubator to reduce condensation and enhance the yield of the first strand cDNA. Place the tube on ice to terminate first-strand synthesis.
d. Second Strand cDNA S,vnthesis 10 Combine the following components in the reaction tube from above:
48.4 ltl Sterile HZO, 16 ~1 SX Second-Strand Buffer, 1.6 pl dNTP Mix (10 mM), 4 ~.120X Second-Strand Enzyme Cocktail for an 80 ~.l total volume. Mix the contents thoroughly with gentle pipetting and spin the tube briefly in a microcentrifuge. Incubate the mixture at 16°C for 1.5 hr. then add 2 ~1 (10 15 units) of T4 DNA Polymerise, mix thoroughly with gentle pipetting and incubate the mixture at 16°C for 45 min. Add 4 ~1 of the EDTA/Glycogen mix to terminate second-strand synthesis. Extract the mixture with an equal volume of buffer-saturated (pH 7.5) phenol:chloroform:isoamyl alcohol (25:24:1). Mix the contents thoroughly by vortexing and spin the tube in a microcentrifuge at 20 maximum speed (up to 14,000 rpm or 13000xg), 4°C for 10 min. to separate layers. Carefully transfer the top aqueous layer to a clean 0.5-ml tube.
Extract the aqueous layer with 100 ~,1 of chloroform:isoamyl alcohol (24:1), vortex, and spin the tube as before to separate the layers. Collect the top layer into a clean 0.5-ml microcentrifuge tube. Ethanol precipitate the double-stranded cDNA by 25 adding one-half volume of 4 M Ammonium Acetate and 2.5 volumes of room-temperature 95% ethanol. Mix thoroughly by vortexing and spin the tube immediately in a microcentrifuge at top speed, room temperature for 20 min.
Remove the supernatant carefully and wash the pellet with 300 p,l of 80%
ethanol. Spin the tube as before for 10 min. and carefully remove the supernatant. Air dry the pellet for up to 10 min. and dissolve the cDNA in 10 ~,1 of sterile H20 and store at -20°C. Analyze the yield and size of cDNA
by running 2 ~.1 of the cDNA solution on a I .2% agarose/EtBr gel with suitable DNA size markers (for example the 1 Kbp ladder, GibcoBRL). If EtBr staining 5 does not show a signal and [a-'2P)dCTP was included in the reaction, dry the agarose gel on a vacuum gel drying system and expose an x-ray film to the geI
overnight at -70°C.
e. d for Lig ion Combine these reagents in a 0.5-ml micmcentrifuge test tube, at room temperature, and in the following order: 5 ~1 ds cDNA, 2 ~,1 Marathon cDNA
Adaptor (10 ~.M), 2 ~.l SX DNA Ligation Buffer, 1 ~.1 T4 DNA Ligase (1 unit/~,1) for a 10 ~1 final voluu~e. Mix the contents thoroughly with gentle pipetting and spin the tube briefly in a microceatrifuge. Incubate at either:
lb°C
overnight; or mom temperature (19-23°C) for 3-4 hr. Inactivate the ligase 15 enzyme by heating the mixture at 70°C for 5 min. Dilute 1 ~1 of this reaction mixture with 250 ~.1 of Tricine-EDTA buffer and use for RACE protocols.
Store the undiluted adaptor-ligated cDNA at -20°C for future use.
f. pig For the initial discovery of NaN, we used generic primers designed 20 against highly conserved sequences in domain 1 (D1) of a-subunits I, II and III
and later added more primers to accommodate the new a-subunits that were discovered. Thus, we used generic primers that recognize conserved sequences in all known Na+ channels. The middle of the amplified region shows significant sequence and length polymorphism (Fig. 6) and [47,48]. Due to 25 codon degeneracy, 4 forward primers (F1-F4) and 3 reverse primers (Rl-R3) were designed to ensure efficient priming from all templates that might have been present in the cDNA pool (Table 1 ); however, any of these primers may bind to multiple templates depending on the stringency of the reaction.
Forward primer F1 matches subunits aI, aIII; aNab; aPNl; ail, arHl and aSNS/PN3.
Sequences of individual subunits show 1 or 2 mismatches to this primer: T to C
at position 16 and A to' G at position 18 (aNa6); C to R at position 6 (a~.l);
A
to G at position 18 (arHl) and T to C at position 3 (aSNS). Forward primer F2 matches subunit aII. Forward primer F3 perfectly matches aNa6 and also matches arHl with a single mismatch of C to T at position 16. Reverse primer Rl matches subunits aI, aII, aIII, aNa6, aPNl, a~,l and arHl. This primer has mismatches compared to 4 subunits: G to A at position 3, A to G at position 4 and T to G at position 7 (al); T to C at position 1 and A to G at 10 position 19 (aPNl); G to A at position 3 and A~to G at position 7 (a~.l);
an extra G after position 3, GC to CT at positions 14-15, and A to T at position (arHl). Reverse primer R2 matches subunit aSNS/PN3.

WO 99!38889 PCT/US991~008 Table 1: Generic and NaN specific primers used for the ideatification and cloning of NaN. All primers except the marathon primers, were synthesized at the department of Pathology, Frogratn for Critical Technologies in Molecular Medicine, Yale University.
S Forward Primers Reverse Primers .__~-.

1. GACCCRTGGAATTGGTTGGA 1. CAAGAAGGCCCAGCTGAAGGTGTC

2. AATCCCTGGAATTGGTTGGA 2. GAGGAATGCCCACGCAAAGGAATC

3. GACCCGTGGAACTGGTTAGA 3.
AAGAAGGGACCAGCCAAAGTTGTC

4. GATCTTTGGAACTGGCTTGA 4. ACYTCCATRCANWCCCACAT

S. AACATAGTGCTGGAGTTCAGG S. AGRAARTCNAGCCARCACCA

6. GTGGCCTTTGGATTCCGGAGG 6. TCTGCTGCCGAGCCAGGTA

7. CTGAGATAACTGAAATCGCC

M~thon AP-1 CCATCCTAATACGACTCACTATAGGGC

Marathon AP-2 ACTCACTATAGGGCTCGAGCGGC

1S We used the respective mouse atypical sodium chamiel mNa,2.3 sequence to design forward primer F4 and reverse primer R3 to amplify the analogous sequence from aNaG, the prestmzed rat homolog of mNa,2.3 [14].
The amplified sequence was cloned into the Srf I situ of the vector pCR SCR~T (Stratagene). The nucleotide sequence of this &~agtnent shows 88% ide~ity to the respective sequence of mNa,2.3 (Dib-Ha~j and Waxman, unpublished [68]). The restriction enzyme Xba I was found to be unique to this subunit. Recently, the sequence of a full length cDNA clone of putative sodium channel, NaG-like (SCL-11:Y09164), subunit was published [S]. The published sequence is 99'/o identical to our sequence and confirms the size and restriction 2S enzyme polymorphism of the NaG PCR product.

The predicted lengths of amplified products and subunit-specific restriction enzyme recognition sites are shown in Fig. 6. All subunit sequences are based on Genbank database (accession numbers: aI: X03638; aII: X03639;
aIII: Y00766; aNa6: L39018; ahNE Na: X82835; ap.l M26643; arHl M27902 and aSNS X92184; mNa 2.3 L36719).
Subsequently, amplification of NaN sequences 3' terminal to the aforementioned fi~agment was achieved using NaN-specific primers and two generic reverse primers, R4 and R5. The sequence of the R4 primer was based on the amino acid sequence MWV/DCMEV located just N-terminal to domain 10 II S6 segment (see schematic diagram of Fig. 3 of voltage-gated sodium channel oc-subunits for reference). The sequence of the RS primer is based on the amino acid sequence AWCWLDFL which forms the N-terminal portion of domain III
S3 segment.
Amplification was typically performed in 60 ~,1 volume using 1 pl of the first strand cDNA, 0.8mM of each primer and 1.75 units of Expand Long Template DNA polymerise enzyme mixture (Boehringer Mannheim).
Compared to conventional and thermostable DNA polymerises, Expand Long Template enzyme mixture increases the yield of the PCR products without an increase in non-specific amplification [49,50]. The PCR reaction buffer consisted of 50 mM Tris-HCl (pH 9.2), 16 mM (NH4)ZS04, 2.25 mM MgCl2, 2% (v/v) DMSO and 0.1 % Tween 20. As described previously [46], amplification was carried out in two stages using a programmable thermal cycler {PTC-200, MJ Research, Cambridge, MA.). First, a denaturation step at 94°C for 4 min, an annealing step at 60°C for 2 min and an elongation step at 25 72°C for 90 sec. Second, a denaturation step at 94°C for 1 min, an annealing step at 60°C for 1 min and an elongation step at 72°C for 90 sec. The second stage was repeated 33 times for a total of 35 cycles, with the elongation step in the last cycle extended to 10 min.
Primary RACE amplification was performed in 50 ~.l final volume using 4 ~,1 diluted DRG marathon cDNA template, 0.2 ~,M marathon AP-l and NaN-specific primers, 3.5 U Expand Long Template enzyme mixture.
Extension period was adjusted at 1 min/800 by based on the expected product.
5' and 3' RACE amplification was performed using primer pairs marathon AP-1/NaN specific R6 and NaN specific FS/marathon AP-1, respectively. The 5 PCR reaction buffer consisted of SO mM Tris-HCl (pH 9.2), 16 mM (NH4),SO"
3.0 mM MgCI" 2% (v/v) DMSO and 0.1 % Tween 20. Amplification in three stages was performed in a programmable thermal cycler (PTC-200, MJ
Research, Cambridge, MA.). An initial denaturation step at 92~C was carried out for 2 min. This was followed by 35 cycles consisting of denaturation at 92~C for 20 sec; annealing step at 60~C for 1 min, and an elongation step at 68~C.
Finally, an elongation step at 68~C was carried out for S min. Nested amplification was performed using 2 p,l of a 1/500 diluted primary RACE
product in a final volume of 50 ~,1 under similar conditions to the primary RACE reactions. Primer pairs AP-2/NaN-specific R7 and NaN-specific 15 F6/marathon AP-2 were used for nested 5' and 3' RACE, respectively.
Secondary RACE products were band isolated from 1 % agarose gels and purified using Qiaex gel extraction kit (Qiagen Inc.).
A schematic diagram of the putative structure of NaN is shown in Fig.3.
The length of the intracellular loops is highly variable both in sequence and 20 length among the various subunits. The exception is the loop between domains III and IV.
Example 2: petermination of the Putative Rat ~nino Acid Sequence for the NaN Chi NaN related clones and secondary RACE fi~agments were sequenced at the 25 W. M. Keck Foundation Biotechnology Resource Lab, DNA sequencing group at Yale University. Sequence analysis including determination of the predicted amino acid sequence was performed using commercial softwares, Lasergene (DNASTAR) and GCG, Inc. The putative amino acid sequence of NaN is shown in Fig. 2. Predicted transmembrane segments of domains I - IV are underlined.
Example 3:
Total RNA extraction from trigeminal ganglia of mice, purification of polyA+ RNA, and Marathon cDNA construction were done as previously described for the rat. The initial amplification was performed using rat NaN
primers. The forward primer corresponds to nucleotides 765-787 of the rat sequence (5' CCCTGCTGCGCTCGGTGAAGAAG 3'), and the reverse primer corresponds to nucleotides 1156-1137 (negative strand) of the rat sequence (5' GACAAAGTAGATCCCAGAGG 3'). The amplification produced a fragment of the expected size. The sequence of this fragment demonstrated high similarity to rat NaN. Other fragments were amplified using different rat primers and primers designed based on the new mouse NaN sequence that was being produced. Finally, longer fragments were amplified using mouse 15 Marathon cDNA template and mouse NaN specific primers in combination with adaptor primers that were introduced during the Marathon cDNA synthesis.
These fragments were sequenced using primer walking and assembled into Figure 7A.
Mouse NaN nucleotide sequence, like rat NaN, lacks the out-of frame ATG at the -8 position relative to the translation initiation colon ATG at position 41 (Fig.7A). Translation termination colon TGA is at position 5314. A
polyadenylation signal (AATAAA) is present at position 5789 and a putative 23 nucleotide polyA tail is present beginning at position 5800. The sequence encodes an ORF of 1765 a.a. (Fig. 7B), which is 90% similar to rat NaN. The gene encoding NaN has been named Scnl la.
Chromosomal localization of mouse Nan A genetic polymorphism between strains C57BL/6J and SPRETBi was identified by SSCP analysis of a 274 by fragment from the 3'LTTR of Scnll a.

Genotyping of 94 animals from the BSS backcross panel (Rowe et al., 1994) demonstrated linkage of Scnll a with markers on distal chromosome 9 (Fig. 10).
No recombinants were observed between Scnlla and the microsatellite marker D9Mitl9. Comparison of our data with the MGD consensus map of mouse 5 chromosome 9 revealed close linkage of Scnll a with the two other TTX-R
voltage-gated sodium channels, ScnSa (George et al., 1995; Klocke et al., 1992) and ScnlOa (Kozak and Sangameswaran, 1996; Souslova et al., 1997).
Example 4: Determination of a Partial Humor NaN Se~quence\
Human DRG tissue was obtained from a transplant donor. Total RNA
10 extraction and cDNA synthesis were performed as described previously.
Forward primer corresponds to s~uence 310-294 (minus strand) of EST
AA446878. The sequence of the primer is 5' CTCAGTAGTTGGCATGC 3'.
Reverse primer corresponds to sequence 270-247 (minus strand) of EST
AA88521 1. The sequence of the primer is 15 5'GGAAAGAAGCACGACCACACAGTC 3'. Amplification was performed as previously described. PCR amplification was successful and a 2.1 Kbp fragment was obtained. This fragment was gel purified and sent for sequencing by primer walking, similar to what is done for mouse NaN. The sequence of the ESTs is extended in both directions; the additional sequence shows highest similarity to 20 rat and mouse NaN, compared to the rest of the subunits.
The sequence of a human 2. 1 kbp fragment was obtained using the PCR
forward and reverse primers for sequencing from both ends of the fragment.
Two additional primers were used to cover the rest of the s~uence. The sequence was then extended in the 5' direction using forward primer 1 (above) 25 and human NaN reverse primer (5'-GTGCCGTAAACATGAGACTGTCG3') near the 5' end of the 2.1 kb fragment. The partial amino acid sequence is set forth in Figure 8B.
The partial ORF of the human NaN consists 1241 amino acids. The sequence is 64% identical to the corresponding sequence of rat NaN (73%

similar, allowing for conservative substitutions) using the advanced BLAST
pmgram at NIH. Using the Clustal method of alignment (Lasergene software, DNAStar, Inc.) the human NaN is 68% and 69% similar to mouse and rat NaN, respectively. The respective mouse and rat sequences are 88% similar.
Example 5: Isolation of an Alternative S,~,g Variant of Rat NaN
A rat NaN cDNA that encodes a C-terminal truncated version of the full-length rat NaN in Figures 1 and 2 was isolated by sequencing the insert of a rat cDNA clone. The variant NaN cDNA encodes an NaN protein lacking the 387 C-terminal amino acids of the full length NaN and containing a novel 94 amino acid stretch at the C-terminal end. The new sequence arises from the use of a cryptic donor splice site in exon 23 and a novel exon 23', which is located in intron 23. The novel C terminal amino acids are: AAGQAMRKQG
DILGPNIHQF SQSSETPFLG CPQQRTCVSF VRPQRVLRVP
WFPAWRTVTF LSRPRSSESS AWLGLVESSG WSGLPGESGP SSLL. The N-terminal amino acids of the truncated variant are identical to amino acids 1-1378 of the full length rat NaN of Figure 2. The alternative exon and the splicing pattern was confirmed by comparing the cDNA sequence and the genomic sequence in the respective region.
Example 6: Methods to Isolate Other NaN Seouences a. Isolation off NaN seauences om genomic DNA
The genomic structure of 3 voltage-gated Na+ channel a-subunits have akeady been determined [51-54J. These genes bear remarkable similarity in their organization and provide a predictable map of most of the exon/intron boundaries. Based on the available rat, mouse and human cDNA sequence of NaN, disclosed herein, PCR primers are designed to amplify NaN homologous sequences from other species using standard PCR protocols.
RECTIFfED Si~IEET (RULE 91y Alternatively, commercially available genomic DNA libraries are screened with NaN specific probes (based on the rat, mouse, or more preferably, the human sequence) using standard library screening procedures [59, 60]. This strategy yields genomic DNA isolates that can then be sequenced and the exon/intron boundaries determined by homology to the rat, mouse or human cDNA sequence.
b. Col t~of full length NaNse~Quences,from human auto~v or b.l. Isolation o, human ~an~ia total RNA
A full length NaN human cDNA homologue is isolated from human dorsal root ganglia or trigeminal ganglia or other cranial ganglia from post-mortem human material, foetuses or biopsy or surgical tissues. Total ribonucleic acid (RNA) is isolated from these tissues by extraction in guanidinium isothiocyanate [69] as described in Example 1.
b.2 Determination o, the 1~~ 1 length transcri~ size ,f the human homolog~~fthe ra_t NaNsodium channel cDNA.
The method of determining transcript size is as described in Example 9.
Example 7: Production of human DRG cDNA library A cDNA library from human DRG or trigeminal ganglia polyA+ RNA
was prepared in Example 4 using standard molecular biology techniques [59, 60].
PolyA+ mRNA is hybridized to an oligo(dT) primer and the RNA is copied by reverse transcriptase into single strand cDNA. Then, the RNA in the RNA-DNA hybrid is fragmented by RNase H as E. coli DNA polymerase I
synthesizes the second-strand fragment. The ends of the double sanded cDNA
are repaired, linkers carrying specific restriction enzyme site (for example, Eco wo ~r~ssss rc~r~s~rozoos RI) are ligated to the ends using E. coli DNA ligase. The pool of the cDNA
insert is then ligated into one of a variety of bacteriophage vectors that are commercially available like Lambda-Zap (Stratagene). The procedures are summarized in more detail as follows:
a. First strand cDNA ~~vnthesis Dissolve 10 p,g poly(A) + RNA at a concentration of 1 ~eg/~1 in sterile water. Heat the RNA for 2-5 min. at 65°C-70°C, then quench immediately on ice. In a separate tube add in the following order (180 wl total) : 20 ~1 5 mM
dNTPs (500 uM final each), 40 p.l 5x RT buffer (lx final), 10 p,l 200 mM DTT
IO (10 mM final), 20 pl 0.5 mglml oligo (dT)12-18 (50 ~g/ml final), 60 ~l HZO, p,l (10 U) RNasin (50 U/ml final). Mix by vortexing, briefly microcentrifuge, and add the mixture to the tube containing the RNA. Add 20 p,l (200 U) AMV or MMLV reverse transcriptase for a final concentration of 1000 U/ml in 200 p.l . Mix by pipetting up and down several times and remove 10 p.l to a separate tube containing 1 p,l of a'iP dCTP. Typically, incubate both tubes at room temperature for 5 min., then place both tubes at 42°C for 1.5 hr. This radiolabeled aliquot is removed to determine incorporation and permit an estimation of recovery; this reaction is stopped by adding 1 p,1 of 0.5 M
EDTA, pH 8.0, and stored frozen at -20°C. The radiolabeled reaction will be used later to estimate the yield and average size of the cDNA inserts. The main reaction is stopped by adding 4 p, l of 0.5 M EDTA, pH 8.0, and 200 p 1 buffered phenol. The mixture is vortexed well, microcentrifuged at room temperature for 1 min. to separate phases, and the upper aqueous layer is transferred to a fi~esh tube. Back extract the phenol layer with 1X TE buffer (10 mM Tris, 1 mM EDTA, pH 7.5) and pool the aqueous layers from the two extractions. This back extraction of the phenol layer improves the yield. The cDNA is ethanol precipitated using 7.5 M ammonium acetate (final concentration 2.0 to 2.5 M) and 95% ethanol. Place in dry ice/ethanol bath 15 min., warm to 4°C, and microcentrifuge at 10 min. at full speed, 4°C, to pellet nucleic acids. The small, yellow-white pellet is then washed with ice-cold 70%
ethanol, and microcentrifuged for 3 min. at full speed, 4°C. Again, remove the supernatant, then briefly dry the pellet.
b. second strand s, tvn hesis 5 Typically, the pellet from the first-strand synthesis is resuspended in 284 wl water and these reagents are added in the following order (400 p,l total):

p.l 5 mM dNTPs (50 uM final each), 80 p.l Sx second-strand buffer (lx final), 12 p.l 5 mM ~i-NAD (150 uM final), 2 ~,1 10 uCi/~,1 a 32P dCTP (50 uCi/ml final). Mix by vortexing, briefly microcentrifuge, and add: 4 p.l (4 U) RNase H
(10 U/ml final), 4 p.l (20 U) E. coli DNA ligase (50 U/ml final), and 10 ~,1 (100 U) E. coli DNA polymerase I (250 U/ml final). Mix by pipetting up and down, briefly microcentrifuge, and incubate 12 to 16 hr at 14°C. After second-strand synthesis, remove 4 pl of the reaction to determine the yield from the incorporation of the radiolabel into acid-insoluble material. Extract the second-15 strand synthesis reaction with 400 p.l buffered phenol and back extract the phenol phase with 200 p 1 TE buffer, pH 7.5, as described above. The double stranded cDNA is then ethanol precipitated as described above.
To complete the second-strand synthesis the double-stranded cDNA
ends are rendered blunt using a mixture of enzymes. Resuspend the pellet in 42 p,l water then add these reagents in the following order (80 pl total) : 5 ~1 mM dNTPs (310 uM final each), 16 ~,1 Sx TA butler (lx final), 1 pl 5 mM ~i-NAD (62 uM final): Mix by vortexing, microcentrifuge briefly, and add: 4 It 1 of 2 ~.g/ml RNase A (100 ng/ml final), 4 p.l (4 U) RNase H (50 U/ml final), 4 wl (20 U) E. coli DNA ligase (250 U/ml final), and 4 ~,1 (8 U) T4 DNA
polymerase (100 U/ml final). Mix as above and incubate 45 min at 37°C.
Add 120 p 1 TE buger, pH 7.5, and 1 p.l of 10 mg/ml tRNA. Extract with 200 ~ 1 buffered phenol and back extract the phenol layer with 100 p.l TE buffer as described above. Pool the two aqueous layers and ethanol precipitate as described above.

c. Addition oflinkers to double stranded cDNA
Combine these reagents in a 0.5-ml microcentrifuge test tube, at room temperature, and in the following order: 100 ng ds cDNA, 2 wl linkers/adaptors ( 10 ~, 2 ~,1 5X DNA Ligation Buyer, 1 ~,1 T4 DNA Ligase ( unit/~,1 ) for a 10 ~,1 final volume. Mix the contents thoroughly with gentle pipetting and spin the tube briefly in a microcentrifuge. Incubate at either: 16°C
overnight; or room temperature (19-23°C) for 3-4 hr. Inactivate the ligase enzyme by heating the mixture at 70°C for 5 min. This cDNA is typically digested by Eco RI to prepare the cohesive ends of the cDNA for ligation into the vector and to cleave 10 linker concatemers. Typically this reaction consists of the 10 p 1 of the cDNA, 2 ~,1 of lOX Eco RI buffer (depending on the company of source), 2 pl of Eco RI (10 units/p.l) and sterile Hi0 to a final volume of 20 p,l. The mixture is incubated at 37°C for 2-4 hrs.
d. Size,frac 'ovation o~'cDNA
1 S Size exclusion columns are typically used to remove linker molecules and short cDNA fragments (350 bp). For example, a 1-ml Sepharose CL-4B
column is prepared in a plastic column plugged with a small piece of sterilized glass wool (a 5 ml plastic pipet will work fine). The column is equilibrated with 0.1 M sodium chloride in lx TE (IOmM Tris, 1 mM EDTA, pH 7.5). The 20 cDNA is then loaded onto the column and 200 ~,1 fractions are collected. 2 pl aliquots of each fiaction are analyzed by gel electrophoresis and sutoradiography to determine the peak of cDNA elution. Typically, fractions containing the first half of the peak are pooled and purified by ethanol precipitation and resuspending in 10 ~,1 distilled water.

wo ~r~sss9 rc~r~s~roioos e. Cloning ofcDNA into bacterio~rha~e vector Bacteriophage vectors designed for the cloning and propagation of cDNA are provided ready-digested with Eco RI and with phosphatased ends from commercial sources (e.g., lambda gtl0 from Stratagene). The prepared 5 cDNA is ligated into lambda vectors following manufacturer's instructions.
Ligated vector/cDNA molecules are packaged into phage particles using packaging extracts available commercially.
Example 8: Screening of HunQan cDNA Libra~,y a. Labeling ofcDNA, rf~~"probes), or libra screening 10 An RNA probe is used that recognizes nucleotide sequences that are specific to NaN, such as 1371-1751 of NaN. Other nucleotide sequences can be developed on the basis of the NaN sequence (Fig. 2, 7 and 8) such as nucleotides 765-1160 of the human nucleotide sequence. A Hind IIIlBam HI
fragment of NaN was inserted in pBluescript (SK+) vector (Stratagene). The 15 sequence of the resulting construct was verified by sequencing. The orientation of the insert is such that the 5' and 3' ends of the construct delineated by the Hind III and Bam HI restriction enzyme sites, respectively, are proximal to T7 and T3 RNA polymerise promoters, respectively. Digoxigenin-labeled Sense (linearized at the Hind III site and transcribed by T7 RNA polymerise) and 20 antisense (linearized at the Bam HI site and transcribed by T3 RNA
polymerise) transcripts were prepared in vitro using MEGAscript transcription kit (Ambion) according to manufacturer specifications. Briefly, 1 pg linearized template was transcribed with the respective RNA polymerise in a 20 pl final volume containing the following reagents: 1X enzyme mixture containing the respective 25 RNA polymerise and RNase inhibitor and reaction buffer (Ambion), 7.5 mM
ATP, GTP and CTP nucleotides, 5.625 mM UTP and 1.725 mM Dig-11LTTP
(Boehringer Mannheim). In vitro transcription was carried out at 37°C
for 3 hrs in a water bath. DNA template was removed by adding 1 p,l.of RNase-free DNase I (2U/wl) to each reaction and incubating fiuther at 37°C for 15 min.
The reaction was then stopped by adding 30 pl nuclease-free HZ) and 25 pl of LiCl precipitation solution (7.5 M Lithium Chloride, 50 mM EDTA).
The mixture was incubated at -20°C for 30 min. The RNA transcripts were pelleted in a microfuge at 13000xg, 4°C for 15 min. The supernatant was removed and the pellet washed once with 100 pl of 75% ethanol. The mixture was re-centrifuged at 13000xg;, room temperature for 5 min. The pellet was then air-dried in a closed chamber and subsequently dissolved in 100 ml of RNase-free H20. The transcript yield and integrity were determined by 10 comparison to a control DIG-labeled RNA on agarose-formaldehyde gel as described in the DIG/Genius kit according to manufacturer recommendations (Boehringer Mannheim). Alternatively, a skilled artisan can design radioactive probes for autoradiographic analysis.
Other regions of the rat, mouse or human NaN sodium channel cDNA, 1 S like 3' untranslated sequences, can also be used as probes in a similar fashion for cDNA library screening or Northern blot analysis. Specifically, a probe is made using commercially available kits, such as the Pharmacia oligo labeling kit, or Genius kit (Boehringer Mannheim).
b. cDNA library screening 20 Recombinant plaques containing full length human homologues of the NaN sodium channel are detected using moderate stringency hybridization washes (50-60°C, 5 x SSC, 30 minutes), using non-radioactive (see above) or radiolabeled DNA or cRNA NaN specific probes derived from the 3' untranslated or other regions as described above. Libraries are screened using 25 standard protocols [59, 60] involving the production of nitrocellulose or nylon membrane filters carrying recombinant phages. The recombinant DNA is then hybridized to NaN specific pmbes (see above). Moderate stringency washes are carried out.

Plaques which are positive on duplicate filters (i.e., not artefacts or background) are selected for further purification. One or more rounds of screening after dilution to separate the phage are typically performed.
Resulting plaques are then purified, DNA is extracted and the insert sizes of these clones characterized. The clones are cross-hybridized to each other using standard techniques [59) and distinct positive clones identified.
Typically, overlapping clones that encode the channel are isolated.
Standard cloning techniques are then used to produce a full length cDNA
construct that contains any 5' untranslated sequence, the start codon ATG, the coding sequence, a stop codon and any 3' untranslated sequence, a poly A
consensus sequence and possibly a poly A run. If overlapping clones do not produce sufficient fragments to assemble a full length cDNA clone, alternative methods like RACE (PCR-based) could be used to generate the missing pieces or a full length clone.
c. Characterization of,~j~ human homol~~e ful~ h clone A NaN specific cDNA sequence from the full-length clone is used as a probe in Northern blot analysis to determine the messenger RNA size in human tissue for comparison with the rat and mouse messenger RNA size.
Confirmation of biological activity of the cloned cDNA is carried out using methods similar to those described for the rat NaN.
Example 9: Polymerase chain_ re-~~tion ~$~~~ches to clone Total RNA and poly A+ RNA is isolated from human dorsal root ganglia or trigeminal ganglia or other cranial ganglia from post-mortem human material or foetuses or biopsy/surgical tissues as described above.
Preparation of cDNA and PCR-based methods are then used as described previously in Example 1.

Using degenerate PCR primers derived from the rat NaN specific coding sequence (see Fig. 2), the cDNA is amplified using the polymerise chain reaction [69]. A skilled artisan could utilize the many variables which can be manipulated in a PCR reaction to derive the homologous sequences required.
These include, but are not limited to, varying cycle and step temperatures, cycle and step times, number of cycles, thermostable polymerise, and Mg2+
concentration. A greater specificity can be achieved using nested primers derived from further conserved sequences from the NaN sodium channel.
Amplification is typically performed in 60 ~1 volume using 1 pl of the first strand cDNA, 0.8 mM of each primer and 1.75 units of Expand Long Template DNA polymerise enzyme mixture (Boehringer Mannheim).
Compared to conventional and thermostable DNA polymerises, Expand Long Template enzyme mixture increases the yield of the PCR products without an increase in non-specific amplification [49,50]. The PCR reaction buffer consists of 50 mM Tris-HCl (pH 9.2), 16 mM (NH4)ZS04, 2.25 mM MgCl2, 2%
(v/v) DMSO and 0.1 % Tween 20. As described previously [46], amplification is carried out in two stages using a probable thermal cycler (PTC-200, MJ
Research, Cambridge, MA.). First, a denaturation step at 94°C for 4 min, an annealing step at 60°C for 2 min and an elongation step at 72°C
for 90 sec.
Second, a denaturation step at 94°C for 1 min, an annealing step at 60°C for 1 min and an elongation step at 72°C for 90 sec. The second stage is repeated 33 times for a total of 35 cycles, with the elongation step in the last cycle extended to 10 min. In addition, control reactions are performed alongside the samples.
These should be: 1 ) all components without cDNA, (negative control) and 2) all reaction components with primers for constitutively expressed product, e.g, GAPDH.
The products of the PCR reactions are examined on 1-1.6% (w/v) agarose gels. Bands on the gel (visualized by staining with ethidium bromide and viewing under UV light) representing amplification products of the approximate predicted size are then cut from the gel and the DNA purified.

The resulting DNA may be sequenced directly or is ligated into suitable vectors such as, but not limited to, pCR II (Invitrogen) or pGEMT (Promega).
Clones are then sequenced to identify those containing sequence with similarity to the rat, mouse or partial human NaN sodium channel sequence.
Example 10: Clone anal3r~is Candidate clones from Example 9 are fiuther characterized by conventional techniques. The biological activity of expression products is also confirmed using conventional techniques.
Example 11:
tissues Commercially available human fetal cDNA libraries and/or cDNA pools are screened with NaN specific primers (by PCR) or probes (library screening) using PCR standard PCR protocols and standard library screening procedures as described above.
Example 12: Northern Blot of rat DRG or Trigeminal Neurons with 10-30 p,g total DRG and/or RNA from DRG or trigeminal (for positive tissues) and cerebral hemisphere, cerebellum and liver (for negative tissues) is electrophoresed in denaturing 1 % agarose-formaldehyde gel or agarose-glyoxal gel, and then is transferred to a nylon membrane as described in achieved in multiple steps, as detailed in standard molecular biology manuals [59, 60J.
Radiolabeled (specific activity of >10$ dpm/ug) or Digixoginen-labeled RNA
probes are typically used for Northern analysis. An antisense RNA probe (see Example 20, which describes in situ hybridization with a NaN specific probe) is created by in vitro synthesis from a sense DNA fragment. The membrane carrying the immobilized RNA in wetted with 6x SSC, and the membrane is placed RNA-side-up in a hybridization tube. One ml formamide prehybridization/hybridization solution per 10 cm2 of membrane is added.
Prehybridization and hybridization are usually carried out in glass tubes in a commercial hybridization oven. The tubes are place in a hybridization oven and incubated, with rotation, at 60°C for 15 min to 1 hr. The desired volume of probe is pipeted into the hybridization tube, and the incubation is continued with rotation overnight at 60°C. The probe concentration in the hybridization solution should be 10 ng/ml if the specific activity is 10$ dpm/ug or 2 ng/ml if the specific activity is 109 dpm/ug (use 2-10 ng/ml of Digixogenin labeled probe).
The hybridization solution is poured off and an equal volume of 2x SSC/0.1% SDS is added. Incubation with rotation for 5 min at room temperature is carried out. The wash solution is changed, and this step is repeated. To reduce background, it may be beneficial to double the volume of the wash solutions. The wash solution is replaced with an equal volume of 0.2x SSC/0.1% SDS and the tube is incubated for 5 min with rotation at room temperature. The wash solution is changed and this step is repeated (this is a low-stringency wash). For moderate or high stringency conditions, further washes are done with wash solutions pre-warmed to moderate (42°C) or high (68°C) temperatures. The final wash solution is removed and the membrane rinsed in 2x SSC at room temperature. Autoradiography is then performed for up to 1 week. Alternatively, signal is detected using chemiluminescence technology (Arnersham) if non-radioactive probes are used. The transcript size is calculated from the signal from the gel in comparison with gel molecular weight standard markers.
Example 13: tissue ecific distribution of NaN by RT-PCR
NaN specific forward (5' CCCTGCTGCGCTCGGTGAAGAA 3') and reverse primer (5' GACAAAGTAGATCCCAGAGG 3'), were used in RT-PCR
assays using cDNA template prepared from multiple rat. These primers amplify NaN sequence between nucleotides 765 and 1156 (392 bp) and are NaN specific as judged by lack of similarity to sequences in the database (using programs like BLASTN from the National Library of M~icine). Amplification was typically performed in a 60 lel volume using 1 pl of the first strand of cDNA, 0.8 p,M
of each primer and 1.75 units of Expand Long Template DNA polymerase enzyme mixture (Boehringer Mannheim). Compared to conventional and thermostable DNA polymerases, Expand Long Template enzyme mixture increases the yield of the.PCR products without an increase in non-specific amplification [49, 50].
The PCR reaction buffer consisted of 50 mM Tris-HCl (pH 9.2), l6mM
(NH4)ZSO,, 2.25 mM MgClz, 2% (v/v) DMSO and 0.1% Tween 20. As described previously [71 ], amplification was carried out in two stages using a programmable thermal cycler (PTC-200, MJ Research, Cambridge, MA.). First, a denaturation step is performed at 94°C for 4 min., followed by an annealing step at 60 ° C for 2 min, and then an elongation step at 72 ° C
for 90 sec. Second, a denaturation step is performed at 94°C for 1 min, followed by an annealing 15 step at 60 ° C for 1 min, and then an elongation step at 72 °
C for 90 sec. The second stage was repeated 33 times for a total of 25-35 cycles, with the elongation step in the last cycle extended to 10 min.
Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control to ensure that a lack of NaN signals in different tissues was not 20 due to degraded templates or presence of PCR inhibitors. Rat GAPDH
sequences were co-amplified using primers which amplify a 66 by product that corresponds to nucleotides 328-994 (accession number: M17701). The amplified product spans multiple exon/intron splice sites, based on the structure of the human gene [72]. Dnase I treatment was routinely performed prior to 25 reverse transcription to prevent amplification of GAPDH sequences from genomic processed pseudogenes that may have contaminated the total RNA
preparation [73].
NaN is primarily and preferentially expressed in DRG and trigeminal ganglia neurons. Figure 4 shows the results of screening by RT-PCR for the 30 expression of NaN in various neuronal and non-neuronal tissues. Lanes 1, 2, 4, wo ~r~sss9 rcrms~ro~oos 9 and 16 show a single amplification product co-migrating with the 400 by marker, consistent with NaN specific product of 392 bp. Lanes 1 and 16, 2, 4 and 9 contain products using DRG, cerebral hemisphere, retina; and trigeminal ganglia, respectively. Using this assay, NaN was not detected in cerebellum, optic nerve, spinal cord, sciatic nerve, superior cervical ganglia, skeletal muscle, cardiac muscle, adrenal gland, uterus, liver or kidney (lanes 3, 5-8, and 10-15, respectively). The attenuated NaN signal in cerebral hemisphere and retina, and the absence of this signal in the remaining tissues is not due to degraded RNA
or the presence of PCR inhibitors in the cDNA templates as comparable 10 GAPDH amplification products were obtained in a parallel set of PCR
reaction (data not shown).
Example 14: Transformation of a Host Cell with the NaN Co 'ng Sequence Transformed host cells for the measurement of Na+ current or intracellular Na+ levels are usually prepared by co-transfecting constructs into cells such as HEK293 cells with a fluorescent reporter plasmid (pGreen Lantern-I, Life Technologies, Inc.) using the calcium-phosphate precipitation technique [27]. HEK293 cells are typically grown in high glucose DMEM (Life Technologies, Inc.) supplemented with 10% fetal calf serum (Life Technologies, 20 Inc). After 48 hrs, cells with green fluorescence are selected for recording [28].
For preparation of cell lines continuously expressing recombinant channels, the NaN construct is cloned into other vectors that carry a selectable marker in mammalian cells. Transfections are carried out using the calcium phosphate precipitation technique [27]. Human embryonic kidney (HEK-293), Chinese hamster ovary (CHO) cells, or other suitable cell lines are grown under standard tissue culture conditions in Dulbeccos's modified Eagle's medium supplemented with 10% fetal bovine serum. The calcium phosphate-DNA
mixture is added to the cell culture medium and left for 15-20 hr, after which time the cells are washed with fresh medium. After 48 hrs, antibiotic (G418, Geneticin, Life Technologies) is added to select for cells which have acquired neomycin resistance. After 2-3 weeks in 6418, 10-20 isolated cell colonies are harvested using sterile l Oml pipette dps. Colonies are grown for another 4-7 days, split and subsequently ested for channel expression using whole-cell patch-clamp recording techniques and RT-PCR.
Example 15: Production of NaN specific Antibodies Antibodies specific to the rat, mouse or human NaN are produced with an immunogenic NaN-specific peptide by raising polyclonal antibodies in rabbits. In one example, the peptide CGPNPASNKD~FEKEKDSED (rat amino acids 285-304) was selected because it fits the criteria for immunogenecity and surface accessibility. This peptide sequence does not match any peptide in the public databases. The underlined cysteine (C) residue was changed to Alanine (A) to prevent disulfide bond formation. This amino acid change is not expected to significantly affect the specificity of the antibodies.
Peptide synthesis, rabbit immunization, and affnity purification of the antipeptide antibodies were performed using standard procedures. Purified antibodies were then tested on DRG neurons in culture. Immunostaining procedures using these antibodies before and after blocking with excess peptide were performed according to standard procedures.
DRG neurons after 16-24 h in culture were processed for immunocytochemical detection of NaN pmtein as follows. Coverslips were washed with complete saline solution (137 mM NaCI, 5.3 xnM KCI, 1 ITIM
M902 25 mM sorbitol, 10 mM HEPES, 3 mM CaC 12 pH 7.2), fixed with 4%
paraformaldehyde in 0. 14 M phosphate buffer for 10 min at 4°C, washed with three 5-min with phosphate-buffered saline (PBS), and blocked with PBS
containing 20% normal goat serum, 1 % bovine serum albumin and 0. 1 Triton X- 100 for 15 minutes. The coverslips were incubated in anti-NaN
antibody (1:100 dilution) at 4°C overnight. Following overnight incubation, coverslips were washed extensively in PBS and then incubated with goat anti-rabbit IgG-conjugated to Cy3 (1:3000; Amersham) for 2 h at room temperature.
The coverslips were rinsed with PBS and mounted onto glass slides with Aqua-poly-mount. The neurons were examined with a Leitz Aristoplan light 5 microscope equipped with epifluorescence and images were captured with a Dage DC330T color camera and Scion CG-7 color PCI frame grabber (see Figure 7).
Example 16: N~~ression is altered in a neuro~rain model The CCI model of neuropathic pain (Bennett and Xie) was used to study the plasticity of sodium channel expression in DRG neurons. Twenty two adult, femal Sprague-Dawley rats, weighing 240-260g; were anesthetized with pentobarbital sodium (50 mg/kg ip) and the right sciatic nerve exposed at the mid-thigh. Four chromic gut (4-0) ligatures were tied loosely around the nerve as described by Bennett and Xie (1988) Pain 33, 87-107. The incision site was 15 closed in layers and: a bacteriostatic agent administered intramuscularly.
Previous studies have shown that transection of the sciatic nerve induces dramatic changes in sodium currents of axotomized DRG neurons, which is paralleled by significant changes to transcripts of various sodium channels expressed in these neurons. Sodium currents that are TTX-R and the transcripts of two TTX-R sodium channels (SNS/PN3 and NaN) are significantly attenuated while a rapidly repriming silent TTX-S current emerges and the transcript of a-III sodium channel, which produces a TTX-S current, is up-regulated. We have discovered that CCI-induced changes in DRG neurons, 14 days post-surgery, mirror those of axotomy. Transcripts of NaN and SNS, the two sensory neuron-specific TTX-R channels, are significantly down-regulated as is the TTX-R sodium current, while transcripts of the TTX-S a-III sodium channel are up-regulated, in small diameter DRG neurons. These changes may be partly responsible for making DRG neurons hyperexcitable, that contributes to the hyperalgesia that results from this injury.

Example 17: Assa3rs for agents which modulate the activity of the NaN
channel using hatch clam~,ethods Cells lines expressing the cloned Na'" channel are used to assay for agents which modulate the activity of the NaN channel, e.g., agents which block or inhibit the channel or enhance channel opening. Since the channel activation is voltage dependent, depolarizing conditions may be used for observation of baseline activity that is modified by the agent to be tested. Depolarization may be achieved by any means available, for example, by raising the extracellular potassium ion concentration to about 20 to 40 nM, or by repeated electrical pulses.
The agent to be tested is incubated with HEK 293 or other transformed cells that express the Na+channel [28]. After incubation for a sufficient period of time, the agent induced changes in Na+ channel activity can be measured by patch-clamp methods [29]. Data for these measurements are acquired on a Macintosh Quadra 950, or similar computer, using a program such as Pulse (v 7.52, HEKA, German). Fire-polished electrodes (0.8-1.5 MVI~ are fabricated from capillary glass using a Sutter P-87 pulley or a similar instrument. Cells are usually only considered for analysis if initial seal resistance is <5 Gohm, they have high leakage currents (holding current <0.1 nA at -80 mV), membrane blebs, and an access resistance <5 Mohm. Access resistance is monitored and data is not used if resistance changes occur. Voltage errors are minimized using series resistance compensation and the capacitance artifact will be canceled as necessary using computer-controlled amplifier circuitry or other similar methods.
For comparisons of the voltage dependence of activation and inactivation, cells with a maximum voltage error of <10 mV after coiripensation are usually used. Linear leak subtraction is used for voltage clamp recordings.
Membrane currents are typically filtered at 5 KHz and sampled at 20 KHz. The pipette solution contains a standard solution such as: 140 mM CsF, 2 mM
MgCl2, 1 mM EGTA, and 10 mM Na-HEPES (pH 7.3). The standard bathing solution is a standard solution such as 140 mM NaCI, 3 mM KCI, 2 mM
MgCl2, 1 mM CaCl2, 10 mM HEPES, and 10 mM glucose (pH 7.3).
Tetrodotoxin (TTX)-resistant and TTX-sensitive Na+ currents are measured by exposure to appropriate concentrations of TTX and/or by pre-pulse protocols which distinguish between TTX-sensitive and TTX-resistant currents on the basis of their distinct steady-state inactivation properties [22,55].
Data are collected using standard pulse protocols and are analyzed to measure sodium current properties that include voltage-dependence, steady-state characteristics, kinetics, and re-priming. Measurements of current amplitude and cell capacitance provides an estimate of Na+ current density, thereby permitting comparisons of channel density under different conditions [22,30].
Cells are studied in the current clamp mode to study patterns of spontaneous and evoked action potential generation, threshold for firing, frequency response characteristics, and response to de- and hyperpolarization, and other aspects of 15 electrogenesis [55]. These measurements are carried out both in control cells expressing NaN and in cells expressing NaN that also have been exposed to the agent to be tested.
Example 18: Assays for agents which modulate the activit<r of the NaN
The agent to be tested is incubated with cells exhibiting NaN channel activity. After incubation for a sufficient period of time, the agent inducod changes in Na+ channel are measured by ratiometric imaging of [Na+ ], using SBFI. In this method, cytosolic-free Na+ is measured using an indicator for Na+, such as SBFI (sodium-binding benzofuran isophthalate; [33]) or a similar dye.
Cells are first loaded with the membrane-permeable acetoxymethyl ester form of SBFI (SBFI/AM) or a similar dye (usually dissolved in dimethyl sulfoxide (DMSO) at a stock concentration of 10 mM). Recordings are obtained on the stage of a microscope using a commercially available ratiometric imaging setup (e.g., from Georgia Instruments). Excitation light is provided at appropriate wavelengths (e.g., 340:385 nm). Excitation light is passed to the cells thmugh a dichroic reflector (400 nm) and emitted light above 450 nm was collected.
Fluorescence signals are amplified, e.g., by an image intensifier (GenIISyS) and collected with a CCD camera, or similar device, interfaced to a frame grabber.
5 To account for fluorescence rundown, the fluorescence ratio 340:385 is used to assay cytosolic-free Na+.
Far calibration of SBFI's fluorescence, cells are perfused with calibration solutions containing known Na+ concentrations (typically 0 and 30 mM, or 0, 30, and 50 mM [Na''], and gramicidin and monensin. As reported by Rose and Ransom [34], the 345/390 nm fluorescence ratio of intracellular SBFI
changes monotonically with changes in [Na+ ];. Experiments are repeated on multiple (typically at least 4) different coverslips, providing statistically significant measurements of intracellular sodium in control cells, and in cells exposed to various concentrations of agents that may block, inhibit or enhance 15 the activity of the channel. Use of this method is illustrated in Sontheimer et al.
[32].
Example 19: Assays for agents which modul~.te the activity of the NaN
channel by scin 'gLap~ic im~~g Cells lines expressing the cloned Na+ channel are used to assay for agents which modulate the activity of the NaN channel, e.g., agents which block the channel or enhance channel opening. For example, the agent to be tested is incubated with HEK 293 or other transformed cells that express the Na+ channel [28]. After incubation for a sufficient period of time, the agent induced changes in Na''"~ channel activity are detected by the measurement of Na+ influx by 25 isotopic methods. ZfiTa is a gamma emitter and can be used to measure Na+
flux [31 ) and 86Rb+ can be used to measure Na+/K+ATPase activity which provides a measure of Na channel activity [32] ~Rb+ ions are taken up by the Na''!K+ATPase like K+ ions, but have the advantage of a much longer half life ~~ 42K+ [35). Thus, measurement of the unidirectional ouabain-sensitive 86Rb+

uptake provides a quantitative method for assaying Na+/K+-ATPase activity which follows action potentials.
Following incubation of cell expressing NaN to the isotope, the cellular content of the isotope is measured by liquid scintillation counting or a similar method, and cell protein is determined using a method such as the bicinchoninic acid protein assay [36] following the modifications [37] for cultured cells.
~Na and 86Rb+ fluxes are determined in the presence and absence of agents that may block, inhibit, or enhance Na+. This permits deternunation of the actions of these agents on NaN.
10 Example 20: j~, situ hybridization a. Probes Probes are prepared as described above in Example 5.
b. DRG Neuron Culture Cultures of DRG neurons from adult rats were established as described previously [70]. Briefly, lumbar ganglia (L4, LS) from adult Sprague Dawley female rats were freed from their connective sheaths and incubated sequentially in enzyme solutions containing collagenase and then pepsin. The tissue was triturated in culture medium containing 1:1 Dulbecco's modified Eagle's medium (DMEM) and Hank's F12 medium and 10% fetal calf serum, 1.5 mg/ml trypsin inhibitor, I .5 mg/ml bovine serum albumin, 100 U/ml penicillin and 0.1 mg/ml streptomycin and plated at a density of 500-1000 cells/mmi on polyomithine/laminin coati coverslips. The cells were maintained at 37°C in a humidified 95% air/5% C02 incubator overnight and then processed for in situ hybridization cytochemistry as described previously [56, 57]. Trigeminal ganglia can be cultured by a skilled artisan using similar methods.
c. Tissue Pre~ation Adult female Sprague Dawley rats were deeply anesthetized, e.g., with chloral hydrate and perfused through the heart, first with a phosphate-buffered saline (PBS) solution and then with a 4% paraformaldehyde in 0.14 M
Sorensen's phosphate buffer, pH 7.4, at 4°C. Following perfusion fixation, dorsal root ganglia at levels L4 and LS and trigeminal ganglia were collected and placed in fresh fixative at 4°C. After 2-4 hours, the tissue was transferred to a solution containing 4% paraformaldehyde and 30% sucrose in 0.14 M
phosphate buffer and stored overnight at 4°C. Fifteen ~,m sections were cut and placed on poly-L-lysine-coated slides. The slides were processed for in situ hybridization cytochemistry as previously described [24, 56]. Following in situ hybridization cytochemistry, the slides were dehydrated, cleared and mounted with Permount. The results are shown in Fig. 5.
Sections of DRG hybridized with NaN sense riboprobe showed no specific labeling (panel C, Fig. 5). In DRG (panel A, Fig. 5) and trigeminal (panel B) sections hybridized with a NaN antisense riboprobe, with the NaN
signal present in most small (<30 mm diam.) neurons; in contrast, most large 15 (>30 mm diam.) neurons did not exhibit NaN hybridization signal. Sections of spinal cord, cerebellum and liver hybridized with an antisense NaN ribopmbe showed no specific signal (panels D, E and F respectively).
Example 21: bite Seauencg,~
The following are the marine intronic microsatellite sequences. These microsatellites may be polymorphic in the human SCNl 1 a gene and could be used as markers to screen for mutant alleles that are associated with a disease.
Such screening methods, for instance, hybridization or amplification assays, are readily available. See Sambrook et al. or Ausubel et al.

Intron 4; microsatellite is dTdg AGTTTAATGTTGAGTGAATTGTGGTGGTGATTTCCCACTTGAG
GCCTTTGTGTTAAAGCCCAATGTGTGTGTGTGTGTGTGTGTGTGTGTG
TGTGTGTGTGTGTGTGTGTGTGTGTGGTTGGGGGGTGGTGGCAGAGT
CTGGTATTGGTAAGGTGAGAGCAATCCCAGAACGTCCACCTGCTCTT
CCATTTTATTAATCAGGCAGGCCTCT
Intron 5; microsatellite is dGdTdG (dNdG2)x (XS-30) GTAAGCCACTGGCTCTTAACTAAAATGCTCGTTGGCATTAGAA
CATTTCTGAGCTGGGGTGGTGGTGGTGGTGGTGGTGGTGGTGGTGGT
GGTGGTGGTGGTGGTGGTGGTGATGGTGGTGGTGGAGGTGGNGGTG
GAGGTGGTGGCTGTGGTGGTGGNGGTGGTGGTGGTGGTGGANGTGG
ANGTGGTGGCGTGGTGGTGGNGGTGGTGGTGGAGGTGGTGGCTGTG
GTGGTNGTGGTGGC
Intron 6; microsatellite is dCdA
TGTGCATGC>'TGATTCCCAGCTCCTATGGTCTGATTACTCGGT
CCTTAGGAGCAAGGCCAGACTGTCCACCCTGACACACACACACACA
CACACACACACACACACACACACACACACACACAGTGTAGAGAATT
ACCTCATTCTTGGAGTTTCTCTGGAAAAGGAATGTCTCAAAGCCAAG
TTCACAGAGCAACAGCTG

wo ~r~sss9 rcT~s~rozoos Intmn 8; 5' microsatellite is dTdC followed by a stretch of dT
TGTTAGAAACTCTAAGACAATGAAGCACCATGCTGGAAATAA
GAGCACAAACTCTTTCTTCATGCATTACCCACTGCTTGTGCTTTCACC
TTAGTGCTCGTGCTCTCTCTTTCTCTCTCTCTCTCTCTCTCTCTCTCTC
TCTCTCTCTCTCTCTCTCTCTCTCTCTCTTTTTTTTTTTTTT
Intron 8; 3' microsatellite is dCdA
CACACACACACACACACACACACACACACACACACACACAG
AGAAACACTGTCGCAGTCATACATATAAAGATAAATACATCTTAAA
AAAAGAACCATGTGATTGAGTTATAAAATATTCCAACTTAT
Intron l OB; microsatellite is dCdA followed, three nucleotides downstream by dCdA3 AGGTCATTTCCTCTGCAGTGTGCTTGGCAGGAAAAACTTCCTG
GCTATTCAAGTCAGTGCCCTGCTTGATCATCCATGTATCACACACAC
ACAAAACAAACAAACAAACAAACAAAACCCTGGGGAAGAAGGAAG
AGGTTAAGCACATAGGCAGAGAGCAGCCAGGCTGACTCAGAGCAAA
CACCTGATCATTCTTCCAT

Intron 12; microsatellite is dpydG (dt/dCdG) GTGCTGGGATCAAAGGCGTGCGCCGCCACCACGCCCGGCCCC
TTTTTATGTTTCAAATTTACTTTTATCATGTGCACGTGTGTGGGTGCG
TGCATGTGTGTGCGTGCGTGTGCGTGTGNGTGTGNGTGTGTGTGTGT
S GTGTGTGTGTGTGTGTGTGTG
Intron 14; microsatellite is dCdA
CACACACACACACACACACACACACACACACACACACACACA
CACACACACACACACTTGCATCTTTGAGTTAATTGGATAGGCTGAGT
CTTACACCGGAATCATACTGTTGC
Intmn 1 SA; microsatellige is dCdA
CCAATGAGAGACTCTTGTCTCA,AAAAA.GCCATGGTGTCCAGA
TCCTGAGGAATAACACCTAAGAATGTGCTCTGGCCTGAAAACACAC
ACACACACACACACACACACACACACACACACAGTTTTATTTATTTA
TTTAAAAAAATATGTCTCTAGGCATTGCTGAAATGTCTCCTACAGGA

It should be understood that the foregoing discussion and examples merely present a detailed description of certain preferred embodiments. It wiU
be apparent to those of ordinary skill in the art that various modification and equivalents can be made without departing from the spirit and scope of the invention.

REFERENCES CITED
The following documents were cited and discussed above. These and any other documents referred to in this patent specification are hereby incorporated by reference in their entirety.
[1] Catterall, W.A. (1993) Trends in Neurosciences 16: 500-6.
[2] Isom, L.L., De Jongh, K.S. and Catterall, W.A. (1994) Neuron 12:
1183-94.
[3] Goldin, A.L. (1995) in: Handbook of receptors and channels, pp.
73-100 (North, R.A., Ed.) CRC press, Boca Raton, FL.
[4] Akopian, A.N., Sivilotti, L. and Wood, J.N. (1996) Nature 379:
257-62.
[5] Akopian, A.N., Souslova, V., Sivilotti, L. and Wood, J.N. (1997) FEBS Letters 400: 183-187.
[6] Sangameswaran, L. et al. (1996) J. Biol. Chem. 271: 5953-6.
[7] Beckh, S., Noda, M., Lubbert, H. and Numa, S. (1989) EMBO J. 8:
3611-6.

[8] Mandel, G. (1992) J. Membrane Biology 125: 193-205.

[9] Roden, D.M. and George, A.L., Jr. (1997) Am. J. Physiol. 273:
H511-25.

[10] Ptacek, L.J. (1997) Neuromuscul. Disord. 7: 250-5.

[11] Cannon, S.C. (1997) Neuromuscul. Disord. 7: 241-9.

[12] Cannon, S.C. (1996) Trends Neurosci. 19: 3-10.

[13] Rizzo, M.A., Kocsis, J.D. and Waxman, S.G. (1996) European Neurology 36: 3-12.

[14] Felipe, A., Knittle, T.J., Doyle, K.L. and Tamkun, M.M. (1994) J.
Biol. Chem. 269: 30125-31.

[15] Eccles, J.C., Libet, B. and Young, R.R. (1958) J. Physiol. 143:
11-40.

[16] Gallego, R., Ivorra, I. and Morales, A. (1987) J. Physiol. (Lond) 391:39-56.

[17] Kuno, M. and Llinas, R. (1970) J. Physiol. (bond.) 2I0: 807-821.

[18] Dodge, F.A. and Cooley, J.W. (1973) IBM J. Res. Dev. 17:
219-229.

[19] Titmus, M.J. and Faber, D.S. (1986) J. Neumphysiol. 55:
1440-1454.

[20] Sernagor, E., Yarorn, Y. and Werman, R. (1986) Proc. Natl. Acad.
Sci. (USA) 83: 7966-70.

[21] Rizzo, M.A., Kocsis, J.D. and Waxman, S.G. (1995) Neurobiol.
Dis. 2: 87-96.

[22] Cummins, T.R. and Waxman, S.G. (1997) J. Neurophysiology 17:
3503-3514.

[23] Dib-Hajj, S., Black, J.A., Felts, P. and Waxman, S.G. (1996) Pros.
Natl. Acad. Sci. (USA) 93: 14950-4.

[24] Waxman, S.G., Kocsis, J.D. and Black, J.A. (1994) J.
Neurophysiology 72: 466-70.

[25] Gold, M.S., Reichling, D.B., Shuster, M.J. and Levine, J.D. (1996) Proc. Natl. Acad. Sci. (USA) 93: 1108-12.

[26] England, S., Bevan, S. and Docheriy, R.J. (1996) J. Physiology 495: 429-40.

[27] Ukomadu, C., Zhou, J., Sigworth, F.J. and Agnew, W.S. (1992) Neuron 8: 663-76.

[28] Dib-Ha~j, S.D., Ishikawa, K., Cummins, T.R. and Waxman, S.G.
(1997) FEBS Letters 416: 11-14.

[29] Hamill, O.P., Neher, M.A., Sakmann, B. and Sigworth, F.J. (1981) Pflitgers Arch. 391: 85-100.

[30] Rizzo, M.A., Kocsis, J.D. and Waxman, S.G. (1994) J.
Neurophysiology 72: 2796-815.

[31] Kimelberg, H.K. and Waltz, W. (1988) (Boulton, A., Baker, G.
and Walz, W., Eds.).

[32] Sontheimer, H., Fernandez-Marquee, E.; Ullrich, N., Pappas, C.A.
and Waxman, S.G. (1994) J. Neuroscience 14: 2464-75.

[33] Harootunian, A., Kao, J.P.Y., Ecker, B.K. and Tsien, R.Y. (1989) J. Biol. Chem. 264: 19458-19467.

[34] Rose, C.R. and Ransom, B.R. (1996) J. Physiol. (Lond) 491:
291-305.

[35] Kimelberg, H.K. and Mayhew, E. (1975) J. Biol. Chem. 250:
100-104.

[36] Smith; P.K. et al. (1985) Anal. Biochem. 150: 76-85.

[37] Goldschmidt, R.C. and Kimelberg, H.K. (1989) Analytical Biochemistry 177: 41-45.

[38] Dubner, R. (1994) in: Textbook of Pain (Wall, P.D. and Melzack, R., Eds.) Churchill Livingstone Publishers.

[39] Dubuisson, D. and Dennis, S.G. (1977) Pain 4: 161-74.

[40] Iadamla, M.J., Brady, L.S., Draisci, G. and Dubner, R. (1988) Pain 35: 313-26.

[41] Bennett, G.J. and Xie, Y:K. (1988) Pain 33: 87-107.

[42] Kim, S.H. and Chung, J.M. (1992) Pain 50: 355-63.

[43] Seltzer, Z., Dubner, R. and Shir, Y. (1990) Pain 43: 205-18.

[44] Stys, P.K., Ransom, B.R. and Waxman, S.G. (1992) J.
Neurophysiology 67: 236-40.

[45] Chomczynski, P.a.S., N (1987) Anal. Bioch. 162: 156-159.

[46] Dib-Hajj, S.D., Hinson, A.W., Black, J.A. and Waxman, S.G.
(1996) FEBS Letters 384: 78-82.

[47] Gu, X.Q., Dibhajj, S., Rizzo, M.A. and Waxman, S.G. (1997~T.
Neurophysiology 77: 236-246.

[48] Fjell, J., Dibhajj, S., Fried, K., Black, J.A. and Waxman, S.G.
(1997) Molecular Brain Research 50: 197-204.

[49] Barnes, W.M. (1994) Proc. Natl. Acad. Sci. (USA) 91: 2216-2220.

[50] Cheng, S., Fockler, C. Barnes W. M. and Higuchi, R. (1994) Proc.
Natl. Acad. Sci. (USA) 9I: 5695-5699.
[51 ] George, A.L., Jr., Iyer, G.S., Kleinfield; R., Kallen, R.G. and Barchi, R.L. (1993) Genomics 15: 598-606.
[52] Souslova, V.A., Fox, M., Wood, J.N. and Akopian, A.N. (1997) Genomics 41: 201-209.
[53] McClatchey, A.L, Lin, C.S., Wang, J., Hoffman, E.P., Rojas, C.
and Gusella, J.F. (1992) Hum. Mol. Genet. 1: 521-7.
[54] Wang, Q., Li, Z., Shen, J. and Keating, M.T. (1996) Genomics 34:
9-16.
[55] Sontheimer, H. and Waxman, S.G. (1992) J. Neurophysiology 68:
1041-11.
[56] Black, J.A., Yokoyama, S., Waxman, S.G., Oh, Y., Zur, K.B., Sontheimer, H., Higashida, H. and Ransom, B.R. (1994) Brain Research.
Molecular Brain Research 23: 235-45.
[57] Zur, K.B., Oh, Y., Waxman, S.G. and Black, J.A. (1995) Brain Research. Molecular Brain Research 30: 97-105.
[58] Tanaka et al., unpublished.
[59] Sambrook et al., Molecular Cloning: A Laboratory Approach, Cold Spring Harbor Press; NY, 1989.
[60] Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Co., NY, 1995.
[61] Cohen et al., Proc Acad Sci USA (1972) 69: 2110.
[62] Maniatis et al., Molecular Cloning, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 1982.
[63] Graham et al., Virol. (1973) 52: 456.
[64] Wigler et al., Proc. Natl. Acad. Sci. USA (1979) 76: 1373-76.
[65] Southern, J. Mol. Biol. (1975) 98: 503.
[66] Berent et aL, Biotech (1985) 3: 208.
[67] Rose, et al., J. Neurophysiology, 1997 in press.

[68] Dib-Hajj and Waxman, unpublished.
[69] Saiki et al. (1985) Science 230: 1350.
[70] Rizzo, M.A., Kocsis, J.D., and Waxman, S.G. (1994). Slow sodium conductances of dorsal root ganglion neurons: intraneuronal homogeneity and interneuronal heterogeneity. Journal of Neurophysiology 72, 2796-815.
[71] Dib-Hajj, S.D., Hinson, A.W., Black, J.A., and Waxman, S.G.
( 1996). Sodium channel mRNA in the B 104 neumblastoma cell line. FEBS
Letters 384, 78-82.
[72] Benham, C.D., and Tsien, R.W. (1987). A novel receptor-operated Ca2+-permeable channel activated by ATP in smooth muscle. Nature 328, 275-8.
[73] Ercolani, L., Florence, B., Denaro, M., and Alexander, M. (1988).
Isolation and complete sequence of a functional human glyceraldehyde-3-phosphate dehydmgenase gene. J Biol Chem 263, 15335-41.

Claims (29)

-63-

1. An isolated nucleic acid molecule selected from the group consisting of a nucleic acid molecule comprising the sequence shown in Fig. 1, Fig. 7A or Fig. 8A, a nucleic acid molecule which encodes the amino acid sequence of Figure 2, Figure 7B, Figure 8B or allelic variants of said sequences, and a nucleic acid molecule that hybridizes to one of the foregoing sequences under stringent conditions.

2. The isolated nucleic acid of claim 1, wherein the nucleic acid encodes a voltage gated Na+ channel that is preferentially expressed in dorsal root ganglia or trigeminal ganglia

3. The isolated nucleic acid of claim 2, wherein the nucleic acid encodes the human NaN sodium channel.

4. An expression vector comprising the isolated nucleic acid of any of claims 1 to 3, alone or together with appropriate regulatory and expression control elements.

5. A host cell transformed with the expression vector of claim 4.

6. A Na+ channel encoded by the isolated nucleic acid of any of claims 1 to 3.

7. The Na+ channel of claim 6, having the amino acid sequence of Figure 2.

8. A protein molecule having the amino acid sequence of Fig. 2, Figure 7B, Figure 8B or a peptide fragment thereof.

9. A protein encoded by the isolated nucleic acid of claim 6.

10. A method to identify an agent that modulates the activity of the Na +
channel of claim 6, comprising the steps of bringing the agent into contact with a cell that expresses the Na+ channel on its surface and measuring any resultant changes in the sodium current, resultant change in membrane potential or change in intracellular Na +.

11. The method of claim 10, wherein the measuring step is accomplished with voltage clamp measurements or measurement of membrane potential.

12. The method of claim 10, wherein the measuring step is accomplished by measuring the level of intracellular sodium.

13. The method of claim 10, wherein the measuring step is accomplished by measuring sodium influx.

14. The method of claim 13, wherein the sodium influx is measured using 22Na or 86Rb.

15. A method to identify an agent that modulates the transcription or translation of mRNA encoding the Na + channel of claim 6, comprising the steps of bringing the agent into contact with a cell that expresses the Na + channel on its surface and measuring the resultant level of expression of the Na+
channel.

16. A method to treat pain, paraesthesia and/or hyperexcitability phenomena in an animal or human subject by administering an effective amount of an agent capable of altering Na+ current flow through NaN channels in DRG
or trigeminal neurons.

17. A method to treat pain, paraesthesia and/or hyperexcitability phenomena in an animal or human subject by administering an effective amount of an agent capable of modulating the transcription or translation of mRNA
encoding the Na + channel of claim 6.

18. An isolated nucleic acid that is antisense to the nucleic acid of claim 1 and of sufficient length to modulate the expression of NaN channel in a cell containing the mRNA.

19. A scintigraphic method to image the loci of pain generation or provide a measure of the level of pain associated with DRG or trigeminal neuron mediated hyperexcitability in an animal or human subject by administering labeled monoclonal antibodies or other labeled ligands specific for the NaN Na + channel.

20. A method to identify tissues, cells and cell types that express the NaN sodium channel, comprising the step of detecting NaN on the cell surface or intracellularly.

21. A method to identify tissues, cells and cell types that express NaN
comprising the step of detecting the presence therein of NaN encoding mRNA.

22. A method of producing a transformed cell that expresses an exogenous NaN encoding nucleic acid, comprising the step of transforming the cell with an expression vector comprising the isolated nucleic acid of any of claims 1 to 3, together with appropriate regulatory and expression control elements.

23. An isolated antibody specific for the NaN channel or polypeptide fragment thereof.

24. The isolated antibody of claim 23, wherein the antibody is labeled.

25. A method of producing recombinant NaN protein, comprising the step of culturing the transformed host of claim 5 under conditions in which the NaN sodium channel or protein is expressed, and recovering the NaN protein.

26. A therapeutic composition comprising an effective amount of an agent capable of altering, such as by increasing or decreasing, the rapidly repriming current flow in axotomized, inflamed or otherwise injured DRG
neurons.

27. A method to treat acute pain or acute or chronic neuropathic or inflammatory pain and hyperexcitability phenomena in an animal or a human patient by administering the therapeutic composition of claim 26.

28. A method to screen candidate compounds for use in treating pain and hyperexcitability phenomena by testing their ability to upregulate or downregulate the NaN channel mRNA in axotomized, inflamed or otherwise injured DRG neurons.

29. A method of screening for a mutant NaN allele in a nucleic acid sample comprising the step of hybridizing an intronic microsattelite sequence of Example 20 to the sample under conditions of sufficient stringency to produce a clear signal.