JP2002509860A - Regulation of sodium channels in dorsal root ganglia - Google Patents

Abstract

(57) Abstract A novel tetrodotoxin resistant sodium channel has been disclosed, along with an isolated nucleic acid encoding this receptor. Methods are provided for identifying agents that modulate Na ⁺ current through the receptor, as well as related therapeutic and diagnostic methods.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD OF THE INVENTION

The present invention is directed to screening for the identification of novel tetrodotoxin resistance ant sodium channels and nucleotides associated therewith, and agents that are effective in treating acute or chronic pain or other excitatory hypersensitivity. For the assay. This application is based on U.S. Provisional Application No. 60/072990, filed Jan. 29, 1998, Nov. 20, 1998.
U.S. Provisional Application No. 60/109402, entitled "Modulation of Sodium Channels in Dorsal Root Ganglia," filed on Nov. 20, and "Differential Role of GNCF and NGF in the Maintenance of Two TTX-Res, filed on Nov. 20, 1998.
US provisional application 60 / entitled "istant Sodium Channels in Adult DRG Neurons"
No. 109666, all of which are incorporated herein by reference.

[0002]

[Prior art]

 A. Sodium channels Voltage-gated sodium channels are a type of specialized protein molecule.
Triggers electrical impulses in excitable cells (neurons and muscle fibers)
It functions as a molecular battery for seeding. Voltage-dependent Na from rat brain ⁺ The channel consists of an α subunit (260 KDa) that forms a stoma and two auxiliary subunits that can regulate the properties of the α subunit, β1 (36 KDa) and
It consists of three subunits with β2 (33 KDa). Na current through cell membrane
The α subunit alone is sufficient to create a functional channel that
(References 1 and 2 cited below). In vertebrates, nine distinct α
Subunits have been identified and these are some expanding gene families
(References 3, 4-6 herein)
). In addition, orthologs corresponding to some of them are available in various types of mammals including humans.
Cloned from milk species. Certain α subunits are found in tissues and development
(7, 8). Voltage-gated sodium channel α
Abnormal expression patterns or mutations of subunits are numerous in humans and animals.
Underlying obstacles.

[0003] The voltage-gated sodium channel α subunit is composed of four domains (D1-4) which differ in their internal homology but have the same predicted structure, and these domains are composed of three intracellular loops. (L1-3). These four domains are folded into the cell membrane to form a channel. This channel opens both cytoplasmically and extracellularly through the stoma. The stoma opens and closes depending on the physiological state of the cell membrane.

[0004] Each domain consists of six transmembrane segments (S1-6), which are intracellular and extracellular linkers that woven the protein molecule into the cell membrane. The linker of the S5-S6 segment in these four domains contains a sequence that aligns with the pore of the channel and a subset of highly conserved amino acids. This subset acts as a filter to selectively transport sodium ions through the pores of the channel into the cytoplasm, thereby generating an electric current. The amphipathic S4 segment in each of the four domains is rich in basic residues that are repeated every three amino acids, acts as a voltage sensor, and undergoes morphological changes due to differences in voltage across the cell membrane. Wake up. This change also causes a morphological change in the protein such that pores open to the extracellular Na ⁺ ion gradient.

[0005] In the most known voltage-gated sodium channel α-subunit, within a few milliseconds after opening (activation) of the stoma, the channel closes rapidly and changes to the inactive state (inactive). SNS type channels, on the other hand, slowly deactivate and require larger voltage changes for activation. L3, the loop connecting domains D3 and D4, contains a tripeptide that acts as an intracellular plug that, after activation, closes the stoma and triggers the channel to an inactive state. After inactivation, these channels undergo further morphological changes, regain their own resting state, and are ready for the next activation. This period is called recovery from inactivation (repriming).
Different channels reprime at different rates, and repriming in the SNS is relatively fast.

[0006] The voltage-gated sodium channel family is based on amino acid similarities,
It is further divided into two subfamilies (14). Eight of the nine cloned channels belong to subfamily 1. These channels share many structural features, especially in their S4 cell membrane segments.
However, some of the channels have been shown to have distinct dynamic properties for inactivation and repriming. Only one subfamily 2 channel, also called atypical channel, has been identified in human, rat and mouse tissues. This subfamily is primarily characterized by a reduced number of basic residues in the S4 segment and is therefore predicted to have a different membrane voltage dependence compared to subfamily 1. Subfamily 2
Regarding the physiological function of the channel, its electrophysiological properties have not yet been elucidated and are currently unknown.

[0007] Blocking voltage-gated sodium channels by the neurotoxin tetrodotoxin involves functionally classifying these channels into a sensitive (TTX-S) and a resistant (TTX-R) phenotype. Has been helpful. To date, two TTX-R sex channels in mammals have been identified, one of which is unique to very limited sites in the myocardium and central nervous system, and a second TTX-R SNS, the R sex channel, is restricted to peripheral neurons of the dorsal root and trigeminal ganglia. Particular amino acid residues that confer resistance or sensitivity to TTX have been localized to the ion-selective filter of the channel pore. The SNS channel is described in International Patent Application WO 97/01577.

B. Role of sodium channels in disease states Different isotypes of the Na channel α-subunit have different dynamic and voltage-dependent properties, so the excitatory properties of excitable cells can be determined by the precise nature of the types of channels expressed by the cells. Depends on proper mixing. Mutants of the heart and skeletal muscle α subunit have been shown to cause a number of muscle disorders. Some examples are given below. The change of only one basic amino acid residue in S4 of skeletal muscle channel is
It is sufficient to alter the dynamics of this channel and induce a disease state in many patients. Deletion of a tripeptide in L3 of the myocardial channel (adjacent to the inactivation gate) triggers a cardiac disorder called long-term QT syndrome. A single amino acid change in the S5-S6 linker of domain 1 of Scn8a, a region flanking the pores of the channel, results in the mouse mutant "Jolting". Complete loss of this channel due to other mutations causes motor endplate "med" impairment in mice. This mutation is characterized as a loss of stimulation from motor neurons to the muscles where the nerves are distributed.

C. Sodium channels and pain Axonal injury (damage to nerve fibers, also called axon) causes chronic pain (referred to as neuropathic pain). Numerous studies have shown altered excitability of neuronal bodies and dendrites after axonal injury (15-17), and Na ⁺ channels on neuronal bodies and dendrites following axonal injury Changes in density have been shown to occur (18-20). Also, abnormally mixed expression of sodium channels in neurons can lead to abnormal excitement and contribute to irritability, dysesthesia, or pain.

[0010] In a recent study using rat DRG neurons, we found that TTX
-The expression profile of R- and TTX-S currents, and the number of transcripts of mRNAs encoding channels responsible for these currents in DRG neurons,
Following various disorders, it has been shown to change dramatically (21-23). For example, we found that in identified sensory cutaneous afferent neurons, after axonal injury, the slowly inactivating TTX-R-mediated currents attenuated while at the same time rapidly inactivating TTX-R. It has been shown that S-Na ⁺ current is enhanced (21). We also found that in neurons that respond to painful stimuli (pain), after axonal injury (22), down-regulation of SNS transcription (up-regulation) and concomitant up-regulation of α-III transcription (22). After (up-regulation) and (23), the slowly repriming TTX-S current and TTX-R current died,
It has been shown that rapidly repriming TTX-S currents increase. Slowly increasing concentrations of αI and αII mRNAs have also been associated with axonal injury (24). These changes in sodium channel profiles appear to contribute to abnormal neuronal excitation underlying the neuropathic pain suffered by patients following axonal injury.

[0011] Pain-related inflammation (referred to as inflammatory pain) also causes changes in the sodium current profile in pain-responsive DRG neurons. Inflammatory regulators up-regulate TTX-R currents in small C-type DRG neurons that respond to pain in culture (25, 26). The rapid action of such regulators indicates that their action involves post-translational modification of existing TTX-R sex channels. In addition, the present inventors have now found that TT
It was determined that XR Na ⁺ currents were increased and that SNS transcription in C-type DRG neurons was up-regulated (58). The data herein suggest that altered sodium current profiles contribute to pain caused by inflammation.

[0012]

[Problems to be solved by the invention]

D. Treatment of chronic pain Various types of drugs (anticonvulsants such as phenytoin and carbamazepine, antiarrhythmic agents such as mexicine, local anesthetics such as lidocaine) act on Na ⁺ channels. Since various Na ⁺ channels cause sodium currents with different properties, selectively blocking or activating (or other regulating) specific channel subtypes is expected to have important therapeutic value. You. further,
The selective expression of certain α-subunit isoforms (PN1, SNS, NaN) in certain types of neurons provides a means to selectively alter their properties.

[0013] The DRG pain-responsive neurons are the major source of TTX-R Na ⁺ currents in the PNS. Thus, the Na ⁺ channel that causes TTX-R currents provides a relatively specific target for the manipulation of pain-causing neurons. The molecular structure (SNS) of one TTX-R sex channel of such DRG neurons has been identified previously, but prior to the present invention,
It has not been determined whether other TTX-R sex channels are present in these neurons. If such channels were identified, they would be ideal candidates as target molecules that would be selectively expressed in pain-responsive neurons, and their regulation would be a pain transmission. Can be weakened.

[0014]

[Means for Solving the Problems]

The invention includes an isolated nucleic acid molecule encoding a voltage-gated Na ⁺ channel (NaN channel) that is selectively expressed in the dorsal root or trigeminal ganglia. (In our provisional application 60/072990 prior to the present application,
This NaN channel was called NaX. In a preferred embodiment,
The isolated nucleic acid molecule hybridizes to the sequence shown in FIG. 1, FIG. 7A, FIG. 8A, an allelic variant of this sequence, or to said sequence under stringent conditions. Consists of nucleic acid molecules.

In another embodiment, the invention is directed to an expression vector comprising an isolated nucleic acid molecule encoding a voltage-gated Na ⁺ channel that is selectively expressed in the dorsal root ganglia or trigeminal ganglia. Or an expression vector comprising the above-described isolated nucleic acid molecule and appropriate regulatory and expression control elements. In a preferred embodiment, the expression vector comprises a sequence shown in FIG. 1, FIG. 7A, FIG. 8A, an allelic variant of this sequence, or a single vector having a nucleic acid molecule that hybridizes under stringent conditions to said sequence. Consisting of separated nucleic acid molecules.

In addition, the present invention relates to an isolated nucleic acid molecule encoding a voltage-gated Na ⁺ channel, which is selectively expressed in the dorsal root ganglia or trigeminal ganglia, and suitable regulatory and expression control elements. And host cells transformed with an expression vector consisting of In a preferred embodiment, the expression vector comprises a sequence shown in FIG. 1, FIG. 7A, FIG. 8A, an allelic variant of this sequence, or a single vector having a nucleic acid molecule that hybridizes under stringent conditions to said sequence. Consisting of separated nucleic acid molecules.

[0017] The present invention also relates to the present invention, wherein the dorsal root ganglion or the trigeminal ganglion is selectively expressed.
Contains isolated voltage-gated Na ⁺ channels. In a preferred embodiment, the channel has the amino acid sequence shown in FIG. 2, FIG. 7B, FIG. 8B, or the sequence shown in FIG. 1, FIG. 7A, FIG. Encoded by a nucleic acid molecule that hybridizes under stringent conditions to the above sequences. Peptide fragments of this sequence are also included in the present invention.

Another aspect of the present invention is a method for identifying an agent that modulates the activity of a NaN channel, comprising contacting the agent with a cell expressing a Na ⁺ channel on its surface, Measuring the polarization or the resulting change in sodium current. This measurement step is achieved by voltage clamp measurement, by measuring depolarization and intracellular sodium concentration, or by measuring sodium influx.

Another aspect of the invention is a method of identifying an agent that modulates the transcription or translation of an mRNA encoding a NaN channel. In this method, the drug is coated on its surface with N
contacting cells expressing the a ⁺ channel and measuring the resulting level of sodium channel expression.

[0020] The invention also includes a method for treating pain, paresthesia and irritability in an animal or human subject, the method comprising: It consists in administering an effective amount of an agent that can regulate the flowing Na ⁺ current, for example by suppressing or enhancing it. The method may include administering an effective amount of an agent capable of modulating the transcription or translation of mRNA encoding a NaN channel.

[0021] Another aspect of the invention is an isolated nucleic acid that is antisense to a nucleic acid described above. In a preferred embodiment, the antisense nucleic acid is of sufficient length to regulate the expression of NaN channel mRNA in cells containing the mRNA.

Another aspect of the invention is to provide a measure of the level of pain associated with excitatory nervousness mediated by DRG or trigeminal neurons in an animal or human subject, or Is a scintigraphic method for imaging the gene locus of the occurrence of s. This step involves the use of labeled monoclonal antibodies or other N
administering a labeled ligand specific for the aNNa ⁺ channel.

Another aspect of the invention is a method of identifying a tissue, cell and cell type that expresses a NaN sodium channel. The method comprises detecting NaN at the cell surface, on the way to the cell surface, or in the presence of NaN-encoding mRNA.

Further, the invention includes a method of producing a transformed cell that expresses an exogenous nucleic acid encoding NaN. The method comprises the steps of: isolating a nucleic acid molecule having the sequence shown in FIGS. 1, 7A, 8A, an allelic variant of the sequence, or a nucleic acid molecule that hybridizes to said sequence under stringent conditions. Transforming the cells with a vector comprising the appropriate regulatory and expression control elements. The present invention also includes a method for producing a recombinant NaN protein, comprising the steps of: culturing a transformed host under conditions in which a NaN sodium channel or protein is expressed; and recovering the NaN protein. Become.

[0025] The invention also includes an isolated antibody specific for a NaN channel or a polypeptide fragment thereof. The isolated antibody may be labeled.

[0026] Another aspect of the invention is the rapid recruitment of axons, inflamed, or otherwise damaged DRG neurons, or normal DRG neurons that become excitable at high frequencies. A therapeutic composition comprising an effective amount of an agent capable of reducing the flow of a priming sodium current. The invention also includes a method of treating acute pain, acute or chronic nervous or inflammatory pain, and irritability in an animal or human patient by administering the therapeutic composition described above. .

[0027] The invention also includes a method of screening for candidate compounds for use in treating the symptoms of pain and irritability. This method uses NaN channel mRNA.
Or by testing the ability of said candidate compound to alter protein expression or activity in DRG neurons that are axotomized, inflamed, or otherwise damaged.

[0028]

BEST MODE FOR CARRYING OUT THE INVENTION

The present invention relates to a novel gene discovered by the inventors and named NaN. NaN encodes a previously unidentified protein, referred to herein as NaN. NaN belongs to the α-subunit voltage-gated sodium channel protein family and causes TTX-R sodium currents. Such channels underlie the generation and propagation of impulses in excitable cells such as neurons and muscle fibers. NaN is a novel sodium channel with a sequence that is distinct from other channels identified so far. This selective expression of NaN, expressed in sensory cells but not in other neurons,
This channel is an effective target for diagnostic and / or therapeutic use in connection with acute and / or chronic pain pathologies.

Definitions In this specification, some technical terms and wording are used, which have the following meanings.

The terms “modulate” or “alter” refer to up- or down-regulating the amount and activity of a particular receptor, ligand or current flow. For example, the drug is N
By a ⁺ current inhibition (reduction) or enhanced (increased) may modulate Na ⁺ current. Similarly, the agent may be capable of expressing NaN sodium channels at an expressed level or at an expressed level of NN.
may modulate the activity of the aN channel.

The terms “sodium current” or “Na ⁺ current” refer to cell membranes that occur through channels (specialized proteins) that are selectively permeable to particular ions, in this case sodium ions. Means the flow of sodium ions across.

The phrase “potential dependence” means that an ion channel opens when the cell membrane is in a certain voltage range. Voltage-sensitive sodium channels open when the cell membrane is depolarized. These channels then allow Na ⁺ ions to flow into the cell, further promoting depolarization. This causes the cells to generate electrical impulses (also known as action potentials).

The phrase “rapidly repriming” means that current recovers from inactivation more rapidly than other voltage-gated sodium channel family members.

The term “TTX-R” or “TTX-S” refers to the flow of current across a cell membrane,
Mean resistance or sensitivity to tetrodotoxin (a neurotoxin produced in a particular species) concentration of about 100 nM, respectively.

The term “peripheral nervous system (PNS)” refers to parts of the nervous system other than the brain and spinal cord, such as the spine and its associated dorsal root ganglia (DRG) and ganglia, such as the trigeminal ganglia. And peripheral nerve fibers.

The phrase “suppresses Na ⁺ current” means that the agent reduces current flow as compared to control cells that have not been exposed to it. Preferred inhibitors are
It selectively suppresses such currents without affecting the currents of other sodium channels. Otherwise, the inhibitor suppresses the sodium current through the channel of interest to a much greater extent than the other channels.

The phrase “enhancing the Na ⁺ current” means that the agent enhances the current flow as compared to a control cell that has not been exposed to it. Preferred enhancers are
It selectively increases such currents without affecting the currents of other sodium channels. Otherwise, the enhancer increases the sodium current through the channel of interest to a much greater extent than the other channels.

The term “specifically hybridizes” refers to a nucleic acid encoding a NaN sodium channel, such as the DNA sequence shown in FIG. 1, FIG. 7A or FIG. 8A, under very stringent conditions, or References to nucleic acids that hybridize under moderately stringent conditions.

The phrase “isolated nucleic acid” refers to a nucleic acid that has been substantially purified or separated from nucleic acid residues that encode other peptides. By nucleic acid is meant all forms of DNA and RNA, including cDNA molecules and antisense RNA molecules.

The term “RT-PCR” refers to the amplification step using the polymerase chain reaction (PCR) for a specific cDNA template, which follows the reverse transcription (RT) of RNA using the reverse lipase enzyme. . PCR requires primers that are genetic or gene specific and a thermally stable DNA polymerase, such as Taq DNA polymerase.

The phrase “selectively expressed” means that voltage-gated sodium channels are detected and expressed in higher amounts in certain restricted tissues compared to other tissues. For example, voltage-gated Na ⁺ channels selectively expressed in dorsal root ganglia or trigeminal ganglia are detected in greater amounts in dorsal root ganglia or trigeminal ganglia than in other tissues and cell types. Is done. The amount of voltage-gated Na ⁺ channels can be detected by any available method, including detection of specific RNA amounts and detection of channel proteins using specific antibodies.

Characterization of NaN Sodium Channels The present invention relates to a voltage-gated sodium channel α subunit (NaN) that has not been previously identified. NaN is a TTX-R voltage-gated channel and is predicted to be selectively expressed in sensory neurons entering the body (dorsal root ganglion or DRG) and face (trigeminal ganglion). ing.
The open reading frame, the sequence portion encoding the NaN protein molecule, was determined from different animal species (rat, mouse, human) using its putative amino acid sequence shown in FIGS. 2, 7B, 8B. Have been.

All of the proper landmark sequences for voltage-gated sodium channels are present at the predicted location of NaN, indicating that NaN belongs to the sodium channel family. However, NaNs, unlike all sodium channels that have been identified to date, share less than 53% sequence identity with them. NaN differs from the SNS, which is the Na ⁺ channel subunit identified in the PNS prior to this discovery and is the only other TTX-R. The inventors have identified and cloned NaN without using primers or probes that are SNS based or specific. Furthermore, NaN and SNS
Indicates that its predicted open reading frame (ORF) is only 47%
, Which compares to the limited similarity of NaN to all subfamily 1 members.

The low sequence similarity to the existing α subunit clearly confirms that NaN is a novel gene and not just a mutant of the existing channel. The difference in sequence compared to other voltage-gated channels is that NaN is a novel and previously unidentified prototype, a third type of TTX-type that has different properties compared to SNS. Suggests that it may be an R channel. NaN and SNS present in pain-responsive DRG neurons and trigeminal neurons may respond differently to pharmaceutical intervention. Selective expression of NaN in sensory DRG and trigeminal neurons provides a target that selectively alters the properties of these sensory neurons without affecting other neurons in the brain and spinal cord. Designed to modulate the function of TTX-R currents, characteristic of pain-responsive DRG neurons and contributing to the transmission of pain messages and abnormal patterns of excitement after nerve injury and other pain conditions For a more complete understanding of the effects of the given drugs, it is important to further elucidate the nature of the NaN channel.

NaN Nucleic Acids The nucleic acid molecules of the invention include the nucleotide sequences shown in FIGS. 1, 7A, 8A, as well as the nucleotide sequences encoding the amino acid sequences of FIGS. 2, 7B, 8B. The nucleic acid according to the claims of the present invention comprises a nucleic acid having the nucleotide sequence shown in FIGS. 1, 7A and 8A or a nucleotide sequence encoding the amino acid sequence shown in FIGS. 2, 7B and 8B. Includes nucleic acids that specifically hybridize to nucleic acids. A nucleic acid that specifically hybridizes to a nucleic acid consisting of its nucleotide sequence is stably bound to said nucleic acid under very stringent conditions or under moderately stringent conditions. Very stringent conditions,
Alternatively, moderately stringent conditions are those generally defined and used by Sambrook et al. (59) or Ausubel et al. (60). The exact level of stringency is not critical; rather, conditions should be chosen that provide a clear and detectable signal when selective hybridization has occurred.

Hybridization is a function of sequence identity (homology), G + C content of the sequence, buffer salt content, sequence length and double melting temperature (T [m]) (
See Maniatis et al. (62)). Similarities in sequence length, buffer salt concentration and temperature provide useful variables in assessing sequence identity (homology) by hybridization methods. For example, if there is at least 90% homology, generally hybridization will be performed at 6X diluted from 20X SSC.
It is performed at 68 ° C. in a buffer salt such as SCC (see Sambrook et al. (59
)). If the final Southern blot wash buffer salt is used at a low concentration, eg, 0.1 × SSC, at a high temperature, eg, 68 ° C., the two sequences will form a hybrid duplex. Form. The use of the above hybridization and washing conditions together is defined as being a high stringency or highly stringent condition. Moderately stringent conditions can be used when the two sequences have at least about 80% homology. Here, the hybridization is performed at a temperature of about 50 to 55 ° C. using 6 × SSC. For the final wash, a salt concentration of about 1-3XSSC and about 60-68
A temperature of ° C is used. These hybridization and washing conditions are:
Define moderately stringent conditions.

In particular, specific hybridization occurs under the condition that there is a high degree of complementarity between the nucleic acid comprising the isolated sequence and another nucleic acid. In selective hybridization, generally, complementarity is at least about 70%, 75%, 8%.
0%, 85%, preferably about 90-100%, more preferably about 9%.
5 to 100%.

[0048] The homology or identity used individually can be determined using BLAST (Basic
Local Alignment Search Tool analysis (Karlin et al. Proc. Natl. Acd. Sci. USA 87: 2264-2268 (1990) and Altschul, SFJ Mol. Evol. 36: 290-300 (1993)). And both are incorporated herein by reference). BLAST analysis uses algorithms made for sequence similarity searches and utilized by the programs blastp, blastn, blastx, tblastn and tblastx. BLAS
The approach used by the T program considers similar segments between the sequence in question and the database sequence first, then evaluates the statistical significance of all identified pairs, and finally preselects Only those pairs that satisfy the significance threshold being set. For a discussion of the basic issues in similarity searching of sequence databases, see Altschel et al. (Natu
re Genetics 6: 119-129 (1994)). histogram, description, alignments, expect (eg, statistical significance threshold for reporting matches against database sequences), cutoff,
The search parameters of matrix and filter remain at the default settings. blastp, blastx, tblastn and tblastx
The default score matrix used by is the BLOSUM62 matrix, which is incorporated herein by reference (Henikoff et al. Pr.
oc. Natl. Acad. Sci. USA 89: 10915-10919 (1992)). For blastn, the score matrix is set by the ratio of M (eg, the reward score for a pair of matching residues) and N (eg, the penalty score for a pair of mismatching residues), and the default of M and N Values are 5 and -4 respectively
It is.

[0049] The nucleic acids of the invention can be used in various methods according to the invention. For example, these nucleic acids are used as nucleic acid probes to screen libraries of other cDNA and genomic DNA to select by hybridization other DNA sequences encoding homologous NaN sequences. Contemplated nucleic acid molecules include radioactive nucleotides or non-radioactive methods (eg, biotin).
And there is RNA or DNA labeled. Screening can be performed using a variety of stringencies (common combinations of ionic strength, temperature, and / or the presence of formamide) to isolate near or distantly related homologs. (Through a hybridization Tm operation). These nucleic acids can also be synthesized using the polymerase chain reaction (PCR) method.
It is used to produce primers that amplify DNA or genomic DNA. Further, the nucleic acid sequences of the present invention are used to identify adjacent sequences in the genome, such as, for example, flanking sequences that are regulatory elements of NaN. Also, these nucleic acids are used to produce antisense primers or constructs that can be utilized to regulate the level of NaN gene expression. Its amino acid sequence is specific for NaN, can localize NaN to specific cells,
Used to design and produce antibodies that can modulate channel function.

Host Cells Transformed with Vectors The present invention also comprises a recombinant vector capable of replicating and directing the expression of a nucleic acid encoding a sodium channel in a compatible host cell. For example, inserting a DNA into a vector using an enzyme such as T4 DNA ligase according to the present invention can also be performed by various conventional methods. Such insertion of DNA can be easily performed if the DNA and the desired vector have been cut with the same restriction enzyme, since complementary DNA ends are created. if,
Otherwise, it is necessary to modify the truncated ends created by digesting a single strand of DNA to create blunt (blunt) ends. Alternatively, the same result must be achieved by filling the single strand ends with appropriate DNA. In this way, a blunt end ligation reaction is performed. Alternatively, various desired positions are created by linking nucleotide sequences (linkers) to the ends of the DNA. Such a linker consists of a specific oligonucleotide encoding a restriction site recognition sequence.

[0051] A variety of available vectors and suitable host cells are used (59,60). Commercially available vectors include, for example, New Engla
nd Biolabs, Promega Corp, Stratagene
Includes those available from the Company or other sources.

Transformation of a suitable host cell with an rDNA (recombinant DNA) molecule of the present invention is generally accomplished by well known methods that depend on the type of vector used and the host system. When the RNA is inserted into the frog spores, it expresses the channel. However, expression in mammalian cell lines is preferred. For prokaryotic transformation, electroporation and salting methods are commonly employed (see, for example, Cohen et al., 61, 62). For transformation of vertebrate cells with vectors containing rDNA, electroporation, cationic lipid or salt treatment methods are commonly utilized (63, 64).

[0053] Successfully transformed cells, ie, cells that contain a rDNA molecule of the present invention, can be identified by well-known techniques. For example, cells into which the rDNA of the present invention has been introduced can be cloned to produce single colonies. Cells from these colonies
Harvested and lysed, their DNA content is examined by conventional methods to confirm the presence of rDNA (65, 66). Alternatively, proteins produced by cells are assayed by immunological techniques. If a label such as green fluorescent protein is used in the construction of the recombinant DNA, infected cells can be detected in vivo by sorting the cells by the fluorescence of such cells.

For transient expression of a recombinant channel, transformed host cells for measuring Na ⁺ current or intracellular Na ⁺ levels are generally prepared using calcium phosphate precipitation, The reporter plasmid is prepared by infecting cells, such as HDK293 cells, with the construct. HEK293 cells are grown in DMEM (Life Technologies) with high concentrations of glucose supplemented with 10% fetal bovine serum (Life Technologies). 48
After a time, cells showing green fluorescence are selected for recording (28).

For the preparation of cell lines that constantly express a recombinant channel, the NaN construct is cloned into another vector that has a selectable marker for mammalian cells. Transformation is performed by the calcium phosphate precipitation method (27). Human fetal kidney (HEK
-293) Cells, Chinese hamster ovary (CHO) cells, and other suitable cell lines, are grown under standard culture conditions in DMEM medium supplemented with 10% fetal calf serum. A mixture of calcium phosphate and DNA is added to the cell culture medium, and after 15 to 20 hours, the cells are washed with fresh medium. Forty-eight hours later, antibiotics (G418, Gen.
eticin, Life Technologies). After 2-3 weeks, in a medium supplemented with G418, 10-20 isolated cell colonies are picked up using a sterile 10 ml pipette tip. 4-7 more colonies
Channels are grown for days, stripped, and subsequently tested for channel expression by whole-cell patch-clamp recording and RT-PCR.

[0056] As described by Na ⁺ current method for measuring the Rizzo et al. (30) and Dib-Hajj et al. (28), the Na ⁺ current, measured using the patch clamp technique (29). On a MacIntosh Quadra 950 or similar computer, Pulse (v
v7.52 (manufactured by HEKA, Germany). Generally, heat polished electrodes are made from capillary glass using a Sutter P-87 puller or the like. For very precise analyses, it is generally considered for analysis if the initial seal resistance of the cells is <5 Gohm. Such cells have a large amount of leakage current, cell membrane blebs, and a contact resistance of 5 Mohm or less. Generally, contact resistance is monitored throughout the experiment, and if a change in resistance occurs, the data is not used for analysis. Voltage errors are minimized using series resistance correction, and capacitance artifacts are canceled using computer controlled amplifier circuits or other similar methods. To compare the voltage dependence of activation and inactivation, use cells with a maximum corrected voltage error of ± 10 mV. Normally, linear clamp subtraction is used for voltage clamp recording. The membrane current is generally filtered at 5 KHz,
Used for testing in Hz. Pipettes contain standard solutions (eg, 140 mM C
_{sF, 2mM MgCl 2, 1mM EGTA} , and 10mM Na-HEP ES (pH 7.3 )) are included. A standard extracellular solution is typically 140 n
M NaCl, 3 mM KCl, 2 mM MgCl ₂ , 1 mM CaCl ₂ , 10
mM HEPES, and a solution of 10 mM glucose (pH 7.3).

Voltage-clamp studies on transformed cells or DRG neurons using methods such as the intracellular patch-clamp recording method provide a quantitative measure of sodium current density (number of sodium channels in the cell) and channel Provides physiological properties. These methods of measuring the current flowing through an ion channel, such as a sodium channel, have been described by Rizzo et al. (21). As an alternative, blockade or enhancement of sodium channel function is measured by optical imaging using sodium labeled with a sodium-sensitive dye or isotope. These methods are described in Rose et al. (J. Neurophysiology 1
997 (in press) (67) and by Kimelberg and Walz (31), which measure the increase in intracellular sodium ion concentration that occurs when sodium channels are opened.

[0058] Various drug effects on cells expressing the measure Na ⁺ in intracellular sodium ([Na ^+] _i) were using other ion-sensitive dye that similar SBFI or its class [Na ^+] _i in It can be measured by the comparative image method. This method, as described by Sontheimer et al. (32),
Intracellular free Na ⁺ is measured using an indicator for Na ⁺ such as, for example, SBFI (Sodium-binding benzofuran isophthalate; (33)) or a similar dye. Cells are first isolated in a transmembrane acetoxymethyl ester form of the dye (dimethylsulfoxide (DMSO)).
(Dissolved at a stock concentration of 0 mM). Recordings are taken on a microscope table with a comparative image setup (eg, Georgia Instruments). The excitation light has an appropriate wavelength (for example, 340: 385).
nm). The excitation light is passed through the cells via a reflector (400 nm) and collects fluorescence above 450 nm. The fluorescent signal is amplified, for example, by an image intensifier (GenIISyS) and collected by a CCD camera or similar device connected to a frame grabber. Fluorescence ratio of 340 to account for fluorescence decay
: 385 is used for intracellular free Na ⁺ assay.

After each experiment, conversion solutions containing known Na ⁺ concentrations (typically 0 and 30 mM, or 0, 30, and 50 mM [Na ⁺ ]), and solutions such as gramicidin and monensin The ionophore is poured over one side of the cell and a SBFI fluorescence calibration is performed. As reported by Rose and Ransom, 3 of intracellular SBFI
The 45/390 nm fluorescence ratio changes monotonically with changes in [Na ⁺ ] _i . Generally, the experiment is repeated for a number of (typically at least four) different coverslips.
This provides a statistically significant measure of intracellular sodium in control cells and cells exposed to various concentrations of the drug.

Method for Measuring Na ⁺ Influx by Measuring ²² Na or ⁸⁶ Rb ²² Na is a gamma emitter and is used to measure Na ⁺ influx (31). ⁸⁶ Rb is used to measure Na ⁺ / K ⁺ -ATPase activity (32). ^{The 86} Rb ion, like the K ⁺ ion, is removed by Na ⁺ / K ⁺ -ATPase, but has a much longer half-life than ⁴² K ⁺ (35). Thus, measuring ouabain-sensitive ⁸⁶ Rb unidirectional uptake is another indicator of electrical excitation in neurons, Na ⁺ / K ⁺ -ATP.
Quantitative methods for assaying ase activity are provided. Cells expressing NaN are
After incubation with isotope ²² Na, the intracellular content of the isotope is determined by liquid scintillation counting or a similar method. Intracellular proteins were also analyzed according to the modified for cultured cells described by Goldschmidt and Kimelberg (37), for example, the bicinchoninic acid protein assay (3.
It measures by the method like 6). The flux of ²² Na and ⁸⁶ Rb is measured in the presence or absence of agents that block, suppress or enhance NaN. This establishes the effect of these drugs on NaN.

Methods for Identifying Agents that Modulate NaN-Mediated Current Several approaches have been taken to identify agents capable of modulating (ie, blocking or enhancing) Na ⁺ current through NaN sodium channels. Is used. In general, model cell lines expressing NaN sodium channels are used to identify such agents. Also, several conventional assays are used to measure Na ⁺ current. Such conventional assays include the patch clamp method, the image comparison method of [Na ⁺ ] _i , and the use of ²² Na and ⁸⁶ Rb, as described above.

[0062] In one embodiment of the present invention, to assess the activity of a candidate compound that modulates Na ⁺ current, a drug that is expressing NaN in a suitable transformed host cell is used. Contact. After mixing them or after a suitable culture time,
For the agent to determine whether enhanced or inhibited Na ⁺ current amount, to measure the Na ⁺ current.

[0063] Agents that suppress or enhance the Na ⁺ current are thus identified. A variety of well-known techniques are readily available to those skilled in the art in determining whether a particular drug modulates the amount of Na ⁺ current.

Since Na ⁺ is selectively expressed in pain signaling cells, by measuring the response of drug-treated laboratory animals to acute or chronic pain, the Na ⁺ channel It is also possible to design agents that block, suppress or enhance function. In certain embodiments of this aspect of the invention, the experimental animal, such as a rat, is treated with, for example, an agent that blocks or inhibits (or is believed to block or inhibit) NaN. The response to various pain stimuli is then measured using test methods such as the tail-flick test and the limb withdrawal reflex and compared to untreated control animals. These methods are described in reference (38).
See Chapter 5. In other embodiments of this aspect of the invention, laboratory animals such as rats are locally injected with a pain-causing inflammatory agent such as formalin, Freund's adjuvant (40) or carrageenin, or Perform nerve compression (41, 42) or nerve cross section (43) that causes sexual pain. The animal's response to various common and painful stimuli is then measured. For example, the latency between the warm or heat stimulus and the withdrawal reflex is measured and control animals are compared to animals treated with a drug believed to alter NaN.

Preferred inhibitors and enhancers of NaN are selective for NaNNa ⁺ channels. They may be absolutely selective (such as tetrodotoxin, TTX, which inhibits sodium channels but does not bind to other channels or receptors and does not directly affect other channels or receptors). Alternatively, it may be relatively selective (such as lidocaine, which binds to and blocks some ion channels, but has a preference for sodium channels). Absolute selectivity is not required for the inhibitor or enhancer to work. The ratio of the effect on sodium channels to the effect on other channels or receptors measures one or more effects of the drug on some channels in addition to the target (44).

It is envisaged that the modulating agents of the invention will be, for example, peptides, small molecules, natural and other poisons, vitamin derivatives, and carbohydrates. If you are an expert on that path,
It can be readily appreciated that there is no limit as to the structural properties of the modulators of the present invention. Agents that modulate NaN or the amount of current through it will be revealed by screening molecular libraries. Similarly, natural poisons (specific fish,
(Such as those produced by amphibians and invertebrates). Routinely, such agents are identified by exposing transformed host cells or other cells expressing sodium channels to these agents and measuring the resulting changes in Na ⁺ current. obtain.

Expression, synthesis and purification of the recombinant protein
It can be expressed in a protease-deficient strain such as G-456, and can be purified by a conventional method.

The peptide agents of the present invention can be prepared using standard solid-phase (or liquid-phase) peptide synthesis methods known in the art. In addition, DNAs encoding these peptides are synthesized using commercially available oligonucleotide synthesizers and recombinantly produced using standard recombinant production systems. Production using solid phase peptide synthesis is required if it contains amino acids that are not encoded by the gene.

Antibodies and Immunodetection Another class of agents of the invention are antibodies that are immunoreactive with Na ⁺ channels. These antibodies may be able to block, suppress or enhance current flow through the channel. These antibodies can be obtained by immunizing a suitable animal subject with a peptide containing sites for the antigenic determinant NaN, particularly (but not necessarily) extracellular sites on cells. Can be These immunological agents can also be used in competitive binding studies to identify secondary inhibitors.
These antibodies, if appropriately labeled by conventional methods, can also be used in imaging studies.

Production of Transgenic Animals Transgenic animals containing mutants, knockouts and modified NaN genes are also included in the present invention. Also, transgenic animals in which both the NaN and SNS / PN3 genes have been modified, split, or modified in some way are included in the invention. A transgenic animal is a genetically modified animal that has been obtained by experimental transfer of recombinant, exogenous, or cloned genetic material. Such genetic material is often referred to as a transgene. The nucleic acid sequence of the transgene is in this case in the form of NaN,
The particular nucleic acid sequence is integrated into either a genomic locus not normally found or into a common locus for transgenes. A transgene consists of a nucleic acid sequence derived from a different type of genome than the target animal type or from the same type of genome.

The term “germline transgenic animal” refers to a transgenic animal in which a genetic mutation or genetic information has been introduced into the germline and has gained the ability to transfer the genetic information to progeny. To tell. If, in fact, such offspring possess some or all of the mutation or genetic information, they are also transgenic animals.

The mutation or genetic information is foreign to the species of animal to which the recipient belongs, foreign to a particular individual recipient, or the genetic information is already owned by the recipient . In the last case, the mutated or introduced gene may be expressed differently than the native gene.

The transgenic animals can be used for gene injection into embryonic stem cells, electroporation,
It can be produced by a variety of different methods, including microinjection, gene targeting, and recombinant and retroviral infections (US Pat.
No. 736866; U.S. Pat. No. 5,602,307; Mullins et al. (1993) Hyperte
nsion 22 (4): 630-633; Brenin et al. (1997) Surg.Oncol. 6 (2) 99-110; Tuan
(ed.), Recombinant Gene Expression Protocols, Methods in Molecular Biolo
gy No. 62, Humana Press (1997)).

A number of recombinant or transgenic mice have been produced. Some of these express an activated oncogene sequence (US Pat. No. 47368).
No. 66), which express the cyvirus SV40T-antigen (US Pat. No. 57289).
No. 15), those lacking the expression of interferon regulatory factor-1 (IRF-1) (US Pat. No. 5,731,490), those showing dopaminergic disorders (US Pat. No. 57237).
No. 19), expressing at least one human gene involved in blood pressure control (US Pat. No. 5,731,489), and exhibiting a condition very similar to that of naturally occurring Alzheimer's disease (US Pat. No. 5,720,936) One with poor ability to mediate cell adhesion (US Pat. No. 5,602,307), one with the bovine growth hormone gene (Clutter et al. (1996) Genetics 143 (4): 1753-1760), or That can generate an antibody reaction (McCarthy (1997) The Lancet 349 (9049): 405)
, Is included.

While mice and rats have been selected as animals for most transgenic experiments, it may be preferable or necessary to use other animal species. The transgenic process has also been used successfully in non-rats, including sheep, goats, pigs, dogs, cats, monkeys, chimpanzees, hamsters, rabbits, cattle, and Guinea pigs (Kim et al. 1997) Mol. Reprod. D
ev. 46 (4): 515-526; Houdebine (1995) Reprod. Nutr. Dev. 35 (6): 609-617; Pe
tters (1994) Reprod. Fertil. Dev. 6 (5): 643-645; Schnieke et al. (1997) S
cience 278 (5346): 2130-2133; and Amoah (1997) J. Animal Science 75 (2):
578-585).

The method for introducing a nucleic acid fragment into a mammalian cell suitable for recombination may be any method that is convenient for co-transformation of a large number of nucleic acid molecules. Detailed steps for producing transgenic animals are described in US Pat. No. 5,489,743 and US Pat.
No. 2307, which are readily available to those skilled in the art.

The specific examples set forth below are merely illustrative and do not limit the scope of the invention.

[0078]

【Example】

[0079]

Example 1 Cloning and Analysis of Rat NaN Gene Coding Sequence a. Preparation of RNA Dorsal root ganglia (DRG) of lumbar vertebrae (L4 to L5) from sexually mature Sprague-Dawley rats
) And the total cell RNA is extracted once with guanidine thiocyanate / phenol (
45). For analysis, DRG tissue was simultaneously excised from several animals and used. RNA quality and relative yield were determined by electrophoresis in a 1% agarose gel. Since the dorsal root ganglion of the raw material was obtained in a limited amount of 10 mg on average from 4 individuals, the RNA yield was not quantified. Poly A + RNA was purified from 300 μg of dorsal root ganglion total RNA using a PolyATtract separation system (Promega) according to the manufacturer's instructions. Half of the purified RNA
It was used for preparing marathon cDNA (described later) without quantification.

B. First strand cDNA was synthesized for reverse transcription analysis essentially as previously reported (46).
Briefly, 1 μM random hexamer (Boe) in a final volume of 25 μl
(Hringer Mannheim) and 500 units of superscript
Total RNA was reverse transcribed using II reverse transcriptase (Life Technologies) in the presence of 100 units of a ribonuclease inhibitor (Boehringer Manheim). The reaction buffer was 50 mM Tris-HCl (pH
8.3) consisted of 75 mM KCl, 3 mM MgCl ₂ , 10 mM DTT and 125 μM dNTP. The reaction was allowed to proceed at 37 ° C for 90 minutes, at 42 ° C for 30 minutes, and stopped by heating at 65 ° C for 10 minutes.

C. First-strand cDNA synthesis The experimental protocol for marathon cDNA synthesis was performed according to the manufacturer's manual, as summarized below (all buffers and enzymes were from the manufacturer Clon).
purchased from tech).

In a sterile 0.5-ml microcentrifuge tube, combine the following reagents: 1 μg (1-4 μl) of poly A + RNA sample, 1 μl (10 μM) of cDNA synthesis primer and sterile H ₂ O to make a total volume of 5 μl. Mix. Mix the components and briefly centrifuge the tube in a microcentrifuge. Incubate the mixture at 70 ° C. for 2 minutes, then immediately quench the tube in ice for 2 minutes. Instantly centrifuge tubes (touch spin)
And collect the condensation. The following reagents were added to each reaction tube: 2 μl of 5X first strand buffer, 1 μl of dNTP mix (10 mM), 1 μl of [α- ³² P] dCTP (1 μCi / μl), and 1 μl of AMV reverse transcriptase (20 units / μl). Add to 10 μl volume. Radiolabeled dCTP (for measuring the yield of cDNA synthesis)
It is any, if not used may be replaced with sterile H ₂ O. Mix the contents of the tube by gently pipetting and touch and spin the tube to collect the contents at the bottom. The mixture is kept at 42 ° C. for 1 hour in an incubator to reduce dew condensation to increase the yield of first strand cDNA. Place the tube on ice to stop the first strand synthesis reaction.

D. Second-strand cDNA synthesis A reagent having the following composition was added to the above reaction tube: 48.4 μl of sterile H ₂ O, 16 μl of 5X second strand buffer, 1.6 μl of dNTP mix (10 mM), 2
Add 4 μl of OX Second Strand Enzyme Cocktail to a total volume of 80 μl
l. Mix the components thoroughly by gentle pipetting and centrifuge the tube briefly in a microcentrifuge. After keeping the mixture at 16 ° C. for 1.5 hours,
Add 2 μl (10 units) of DNA polymerase, mix gently by pipetting gently, and incubate mixture at 16 ° C. for 45 minutes. 4 μl of EDTA / glycogen is added and mixed to terminate the second strand reaction. An equal volume of a buffer-saturated phenol (pH 7.5): chloroform: isoamyl alcohol (2
5: 24: 1). The contents are thoroughly agitated by vortexing, and the tube is centrifuged at 4 ° C. for 10 minutes at 4 ° C. in a microcentrifuge at maximum speed (14,000 rpm or 13000 × g) to separate a liquid layer. Carefully transfer the upper aqueous layer to a clean 0.5 ml tube. The aqueous layer was washed with 100 μl of chloroform: isoamyl alcohol (
24: 1), vortex and centrifuge as before to separate the liquid layer. Carefully collect the upper aqueous layer in a clean 0.5 ml tube. 1 / 2M 4M
Ammonium acetate and 2.5 volumes of 95% ethanol were added at room temperature to give a double-stranded c
The DNA is ethanol precipitated. After thorough vortexing, the tubes are immediately spun in a microcentrifuge at full speed for 20 minutes at room temperature. Carefully remove the supernatant and wash the pellet with 300 μl of 80% ethanol. Centrifuge the tube for 10 minutes as before and carefully remove the supernatant. The pellet is air-dried for about 10 minutes, and the cDNA is dissolved in 10 μl of sterile H ₂ O and stored at −20 ° C. 2 μl of the cDNA solution is mixed with an appropriate molecular weight marker DNA (eg, Gibco BR
The yield and size of the cDNA are analyzed by running on a 1.2% EtBr / agarose gel with a 1 Kbp ladder from L Company). If EtBr staining did not show a signal and the reaction mixture contained [α- ³² P] dCTP, the agarose gel was dried using a vacuum gel dryer and then X-ray film at -70 ° C overnight. Exposure.

E. Ligation of Adapter In a 0.5 ml microcentrifuge tube at room temperature, each reagent was placed in the following order:
5 μl of dscDNA, 2 μl of marathon cDNA adapter (10 μM), 2 μl of 5 X DNA ligation buffer, T4 DNA ligase (1 unit / μl)
l) Add 1 μl in this order to make a total volume of 10 μl. After gentle mixing of the components by gentle pipetting, the tubes are briefly spun in a microcentrifuge. 16 ℃
Incubate either overnight or at room temperature (19-23 ° C) for 3-4 hours. Mix 7
Heat at 0 ° C. for 5 minutes to inactivate the ligase enzyme. Add 1 μl of this reaction solution to 250 μl
Dilute with 1 l Tricine-EDTA buffer and use for RACE protocol. Until using undiluted adapter ligated cDNA -2
Store at 0 ° C.

F. In the initial discovery of the PCR NaN gene, universal primers designed against highly conserved sequences in domain 1 (D1) of α-subunits I, II and III were used, Added more primer to accommodate units. Thus, universal primers that recognize conserved sequences in all known Na + channels were used. There is a significant sequence in the center of the amplification region,
Gene length polymorphism (polymorphism) was observed (FIG. 6 and references (47, 48)). All templates (possible in the cDNA pool, depending on codon degeneracy)
Four forward primers (F1 to F4) and three reverse primers (R1 to R3) were designed to perform priming with high efficiency with respect to Table 1). However, depending on the stringency of the reaction, any of these primers can bind to more than one template. Forward primer F1 matches subunits αI, αIII; αNa6; αPN1; αμ1, αrH1, and αSNS / PN3. One or two mismatches with this primer are shown in the sequence of each subunit. That is, T for C at the 16th position, A (αNa6) for G at the 18th position, C (αμ1) for R at the 6th position, and A (αrH1) for G at the 18th position. ), And T (αSNS) for 3rd place C
It is. Forward primer F2 fits subunit αII. The forward primer F3 perfectly matches αNa6, and a single mismatch for αrH1, ie a C match for T at position 16. Reverse primer R1 matches subunits αI, αII, αIII, αNa6, αPN1, αμ1, and αrH1. This primer has the following mismatch sites when compared to the four subunits. That is, G for A at the third position, T for G at the fourth position, and T (αI) for G at the seventh position; T for C at the first position; A for G (αPN1); G for A at position 3 and A (αμ1 for G at position 7)
); Addition of G following G at position 3, GC for CT at positions 14-15 and A (αrH1) for T at position 21. Reverse primer R2 is subunit αS
Matches NS / PN3.

[0086]

[Table 1]

To amplify a similar sequence from αNaG, a putative rat mNa2.3 analog, a forward primer F4 and a reverse primer R3 were designed using the mouse atypical sodium channel mNa2.3. . The amplified sequence was cloned into the SrfI site of a pCR-SCRIPT vector (Stratagene). The nucleotide fragment was 88 to the corresponding mNa2.3 sequence.
% Identity (Dib-Hajj and Waxman, unpublished (68)). The restriction enzyme XbaI was found to be unique (having only one breakpoint) in this subunit. Recently, NaG-like (SCL-
11: Y'09164) The sequence of the full-length cDNA clone of the subunit has been published (5). The reported sequence is 99% identical to our sequence and the NaG PCR
Product size and restriction polymorphisms are confirmed.

FIG. 6 shows the expected size of the amplification product and the subunit-specific restriction enzyme recognition site. All subunit sequences are stored in the Genbank database (accession number: a
I: X03638; αII: X0339; αIII: Y00766; αNa6: L39018; αhNE-Na: X82835; αμ1 M26643; αrH1
M27902 and αSNBS X92184; mNa2.3 L36761
9).

Subsequently, amplification of the 3′-terminal NaN sequence of the above-mentioned fragment was achieved using two NaN-specific primers and two general-purpose primers R4 and R5. The sequence of the R4 primer was based on the amino acid sequence MWV / DCMEV located just N-terminal to the domain IIS6 segment. (See FIG. 3 for the voltage-gated sodium channel α subunit for reference). The sequence of the R5 primer is based on the amino acid sequence AWCWLD FL, which forms part of the N-terminal part of the domain IIIS3 segment.

The amplification reaction is usually carried out in a 60 μl volume, in which 1 μl of the first strand cDNA, 0.1 μl of each primer.
8 mM and Expand Long Template DNA polymerase
Mixing (Boehringer Mannheim) was performed using 17.5 units. The Expand Long Template DNA polymerase mix increases the yield of PCR products without increasing nonspecific amplification compared to conventional thermotolerant DNA polymerases (49,50). The PCR reaction buffer is
It consists of 50 mM Tris-HCl (pH 9.2), 16 mM (NH ₄ ) ₂ SO ₄ , 2.25 mM MgCl ₂ , 2% (v / v) DMSO and 0.1% Tween20. As described in (46) above, the amplification reaction was performed in two steps using a programmable thermal cycler (PTC-200, MJ Research, Cambridge, Mass.). First, the heat denaturation step was at 90 ° C. for 4 minutes, the annealing step was at 60 ° C. for 2 minutes, and the extension reaction step was at 72 ° C. for 90 seconds. Second, a heat denaturation step at 94 ° C. for 1 minute and an annealing step at 60 ° C.
C. for 1 minute and the extension reaction step at 72 ° C. for 90 seconds. The second stage is 33
The number of repeats was extended, and the elongation reaction step in the final cycle was extended to 10 minutes, and 35 cycles were performed in all stages.

The primary RAGE amplification reaction consisted of 4 μl of the DRG diluted marathon cDNA template,
Marathon AP-1 and NaN specific primers 0.2 μM and Expand
Using 3.5 units of Long Template enzyme mix, the total volume was 50 μl. The extension reaction time was adjusted to 800 bp / min based on the expected product. 5 'and 3' RACE amplification was performed with Marathon AP-1 / NaN-
This was performed using a specific R6 primer pair and a NaN-specific F5 / marathon AP-1 primer pair, respectively. PCR reaction buffer is 50 mM T
It consists of ris hydrochloric acid (pH 9.2), 16 mM (NH ₄ ) ₂ SO ₄ , 3.0 mM MgCl ₂ , 2% (v / v) DMSO and 0.1% Tween20. The three-step amplification reaction was performed using a programmed thermal cycler (PTC-200, MJ Research, Cambridge, Mass.). The first heat denaturation step is 9
It was 2 minutes at 2 ° C. Subsequently, thermal denaturation at 92 ° C. for 20 seconds, annealing step 6
The extension reaction step was performed at 0 ° C. for 1 minute at 68 ° C., and the cycle was 35 cycles. Finally, an extension reaction was performed at 68 ° C. for 5 minutes. Nested PCR is performed using the primary RA
Using 2 μl of a 500-fold dilution of the GE amplification product, a total volume of 50 μl was performed under the same conditions as the primary RAGE reaction. Primer pair, AP-2 / NaN specific R7 and N
aN-specific F6 / marathon AP2 was nested 5 'and 3' RA, respectively.
Used for CE reaction. The secondary RAGE product was band isolated from a 1% agarose gel,
Purification was performed using a Qiaex gel extraction kit (Qiagen Inc.).

FIG. 3 is a schematic explanatory view of the estimated structure of NaN. Intracellular loops vary in length and sequence among various subunits. The exception is the loop between domains III and IV.

[0093]

Example 2 Determination of Deduced Amino Acid Sequence of Rat NaN Channel NaN-related clones and secondary RACE fragments were isolated from Yale University DNA Sequencing Group W.C. M. Sequenced at the Keck Foundation for Biotechnology Resources. Sequence analysis, including predicted amino acid sequencing, was performed using the commercial software Lasergene (DNASTAR, Inc) and GCG. FIG. 2 shows the deduced amino acid sequence of NaN. The transmembrane segments of domains I-IV are underlined.

[0094]

Example 3 Determination of Mouse NaN Sequence Extraction of mouse trigeminal ganglion total RNA, purification of poly A + RNA, and construction of marathon cDNA were performed in the same manner as in the above-described experiment in rats. Primary amplification was performed using rat NaN primers. The forward primer was nucleotide 765-787 of the rat sequence (5'CCCTGCTGGCCTCGGTGAAGAAG3 '
) And the reverse primer corresponds to nucleotide 1156-1 of the rat sequence.
137 (5'GACAAAGTAGATCCCAGAGG3 '; negative strand). A fragment of the expected size was obtained by amplification. The sequence of this fragment demonstrated high similarity to rat NaN. Other fragments were amplified using different rat derived primers and newly designed primers based on the new mouse NaN sequence. Finally, a longer fragment was amplified using the mouse marathon cDNA template and mouse NaN-specific primer in combination with the adapter primer introduced during the synthesis of the marathon cDNA. These fragments are used as primers
The result of sequencing by the walking method is shown in FIG. 7A.

Like the rat NaN, the out-of-frame ATG located at position −8 from the translation start codon ATG at position 41 is deleted in the mouse NaN nucleotide sequence as well. The translation stop codon TGA is located at position 5314. Poly A additional signal (AATA
AA) is located at position 5789, and the poly A tail, estimated to be 23 nucleotides in length, starts at position 5800. The sequence encodes an ORF (open reading frame) of 1765 amino acid residues that is 90% similar to rat NaN (FIG. 7B).
. The gene encoding NaN was named Scn11a.

Chromosome Position of Mouse NaN SSCP analysis was performed on a 274 bp fragment obtained from the 3 ′ UTR of Scn11a, and gene polymorphisms between the C57BL / 6J strain and the SPRET / Ei strain were identified. BSS backcross panel method on 94 animals (Rowe et al., 1994)
(Genotype) was determined, and it was proved that the gene was linked to the chromosome 9 distal end marker group (FIG. 10). No recombination was observed between Scn11a and the microsatellite marker D9Mit19. As a result of comparing the MGD consensus map of mouse chromosome 9 with our data, Scn11a
Is the other two TTX-R voltage-gated sodium channels Scn5a (Ge
Orge et al., 1995; Klocke et al., 1992) and Scn10a (Ko).
zak and Sangameswaran, 1996; Souslova et al., 199.
It became clear that the position was close to 7).

[0097]

Example 4 Determination of Human NaN Partial Sequence Human DRG was obtained from a transplant patient. Total RNA extraction and cDNA synthesis were performed as described above.

The forward primer corresponds to sequence 310-294 (minus strand) of EST AA446878. The sequence of the primer was 5 ′ CTCAGTAGTTGGGC
ATGC3 '. The reverse primer was sequence 27 of EST AA88521-1.
0-247 (minus chain). The sequence of the primer is 5'GGAAA
GAAGCACGACCACACAGTC 3 ′. Amplification was performed as described above. PCR amplification was successful and a 2.1 Kbpf fragment was obtained. The fragment was gel purified and outsourced for sequencing by the primer walking method as was done with mouse NaN. When the EST sequence was extended in both directions and compared with the other subunits, the newly obtained sequence had the highest similarity to mouse NaN.

The human 2.1 kbp fragment was sequenced using the forward and reverse primers at both ends of the fragment used as PCR primers. In addition to these, two additional primers were used for sequencing the remaining portion that could not be extended by both primers. Thus, the forward primer 1 (described above) and the human NaN reverse primer corresponding to the vicinity of the 5 ′ end of the present 2.1 kbp fragment (5′GTGCCGGTAA)
ACATGAGACTGTCG3 ') and extended to the 5' upstream sequence. The partial amino acid sequence is submitted as FIG. 8B.

The partial ORF of human NaN consists of 1241 amino acid residues. The sequence is NI
Analysis using the H Advanced BLAST program showed 64% identity to a substantial portion of rat NaN (73% similarity if traditional substitutions were allowed). Using a parallel comparison by the cluster method (Lasergene software, DNAStar), human NaN was converted to mouse and rat N
Shows 68% and 69% similarity to aN, respectively. The significant similarity between mouse and rat is 88%.

[0101]

Example 5 Isolation of Alternative Splicing Mutant of Rat NaN By sequencing the inserted DNA of the rat cDNA clone, the C-terminal truncation of the rat NaN full-length cDNA shown in FIGS. 1 and 2 was shortened. Rat NaN cDNA encoding the type was obtained. The mutant NaNcD
NA encodes NaN with the deletion of the 387 amino acid residue at the C-terminal side of full-length NaN and a new 94-amino acid residue extension at the C-terminus. This new sequence results from a new exon 23 'consisting of a potential splicing donor site within exon 23 and a portion corresponding to the location of intron 23. The new C-terminal amino acid sequence is:
S AWLGLVESSG WSGLPGESGP SSLL. The N-terminal side of the truncated mutant is the same as amino acid residues 1 to 1378 of full length rat NaN shown in FIG. Alternate exons and their splicing patterns were confirmed by comparing the respective regions of the cDNA and chromosomal genomic sequences.

[0102]

Example 6 Isolation of Other NaN Sequences a. Isolation of NaN sequences from genomic DNA The genomic structure of the three voltage-gated sodium channel α subunits has been determined (51-54). These genes have significant similarities in their organization and provide a predictive map of the boundaries of most exon introns.
Based on the NaN rat, mouse and human cDNA sequences disclosed and available herein, standard PCR protocols are used to design PCR primers that amplify NaN cognate sequences from other species.

Alternatively, NaN (based on rat, mouse or more preferably human sequences)
Commercially available genomic DNA libraries are screened with specific probes using standard library screening methods (59, 60). This strategy provides a genomic DNA isolate that can be sequenced, and exon / intron boundaries can be determined by homology to rat, mouse or human cDNA sequences.

B. Isolation of full-length NaN sequences from human dissection, biopsy and surgical tissue b. 1. Isolation of Human Ganglion Total RNA A full length NaN human cDNA homolog is isolated from human dorsal root or trigeminal ganglia or other head and neck ganglia obtained from postmortem human, fetal or biopsy samples or surgical tissues. Total ribonucleic acid (RNA) is isolated from these tissues by extraction into guanidine isothiocyanate as described in Example 1.

B. 2. Size determination of full-length transcript of human homologue of rat NaN sodium channel cDNA The method for determining the size of the transcript is as described in Example 9.

[0106]

Example 7 Preparation of Human DRG cDNA Library In Example 4, a cDNA library was prepared from human DRG or trigeminal ganglion poly A + RNA using standard molecular biology techniques (59, 60).

The poly A + mRNA was hybridized to an oligo (dT) primer and copied as a single-stranded cDNA using reverse transcriptase. Next, a second strand fragment was synthesized by E. coli DNA polymerase I while fragmenting the RNA in the RNA-DNA hybrid with ribonuclease H. Using Escherichia coli DNA ligase, both ends of the double-stranded cDNA were repaired, and a linker having a specific restriction enzyme (eg, EcoRI) recognition site was ligated. Lambda-Zap (
One of various commercially available bacteriophage vectors such as Stratagene)
Connected to one. The operation procedure will be described in detail below.

A. First-strand cDNA synthesis 10 μg of poly A + RNA is dissolved in sterile water to a concentration of 1 μg / μl. RNA
Is heated at 65-70 ° C. for 2-5 minutes and then immediately quenched in ice. In another tube, 20 μl of 5 mM dNTP mix (500 μM final concentration each), 40 μl of 5 × RT buffer (final concentration 1 ×), 10 μl of 200 mM DTT (final concentration 10 mM)
0.5 mg / ml oligo (dT) 12-18 (final concentration 50 μg / ml), 60 mg / ml
Add 10 μl H2O, 10 units RNasin (final concentration 50 units / ml) in this order. Mix with Voltex, briefly centrifuge, and add this mixture to the tube containing RNA. 20 μl of 200 units of AMV or MMLV reverse transcriptase are added so that the final concentration of 200 μl of solution is 1000 units / ml. After mixing by pipetting up and down several times, add 10 μl of α ³² PdCTP
Transfer to another tube containing 1 μl. Usually, both tubes are incubated at room temperature for 5 minutes, then both tubes are incubated at 42 ° C. for 1.5 hours. Radiolabeled aliquots are collected for estimation of uptake and recovery. The reaction is stopped by adding 1 μl of 0.5 M EDTA, pH 8.0, and stored frozen at −20 ° C. The radiolabeled reaction is used later to estimate the average size and yield of the cDNA insert. The main reaction is stopped by adding 4 μl of 0.5 M EDTA, pH 8.0 and 200 μl of phenol with buffer. Vortex the mixture thoroughly, centrifuge at room temperature for 1 minute in a microcentrifuge to separate the liquid phase, and transfer the upper aqueous layer to a new tube. The phenol layer was washed with 1xTE buffer (10 mM Tris, 1 mM EDTA
, PH 7.5) and collect the aqueous layers of the two extractions. Re-extraction of the phenol layer improves yield. The cDNA was converted to 7.5M ammonium acetate (
Ethanol precipitation using final concentration 2.0-2.5M) and 95% ethanol. Place in a dry ice ethanol bath for 15 minutes, warm to 4 ° C, and pellet nucleic acids by centrifugation at 4 ° C for 10 minutes at maximum speed in a microcentrifuge. The small yellow-white pellet is washed with ice-cold 70% ethanol and placed in a microcentrifuge at 4 ° C. at maximum speed for 3 minutes. Remove supernatant again and dry pellet briefly.

B. Second Strand Synthesis Normally, the pellet of the first strand synthesis reaction is resuspended in 284 μl of water and the following reagents are added in the following order (400 μl total): 4 μl of 5 mM dNTP mix (50 μM each final concentration), 4 μl, 5 × second Chain buffer (final concentration 1x) 80 µl, 5 mM β-N
12 μl of AD (150 μM final concentration), 2 μl of 10 μCi / μl α ³² P-dCTP (50 μCi / ml final concentration). After vortexing and brief microcentrifugation, 4 μl of RNaseH 4 units (final concentration 10 units / ml), 4 μl of E. coli DNA ligase 20 units (final concentration 50 units / ml), and 100 units of E. coli DNA polymerase I ( (Final concentration 250 units / ml) 10 μl. Mix by pipetting up and down, briefly centrifuge, and incubate at 14 ° C. for 12-16 hours. After the second strand synthesis, 4 μl of the reaction solution is taken and the yield is determined from the amount of radiolabel incorporated into the acid-insoluble fraction. The second strand synthesis reaction is extracted with 400 μl buffered phenol and re-extracted with 200 μl TE buffer pH 7.5 as described above. Next, the double-stranded cDNA is ethanol precipitated as described above.

To complete second strand synthesis, both ends of the double-stranded cDNA are treated with an enzyme mixture to smooth the ends. Resuspend the pellet in 42 μl water and add the following reagents in the following order (80 μl total): 5 μl 5 mM dNTP mix (310 μM each final concentration) 16 μl, 5 × TA buffer (1 × final concentration) 16 μl, 5 mM βNAD (final concentration 6 μl)
2 μM) 1 μl. Vortex and briefly microcentrifuge and add: 2 μg / ml RNaseA (final concentration 100 ng / ml) 4 μl, RNaseH 4 units (final concentration 50 units / ml) 4 μl, E. coli DNA ligase 20 units (final) 4 μl of a concentration of 250 units / ml) and 4 μl of 8 units of T4 DNA polymerase (final concentration of 100 units / ml). Mix as before and incubate at 37 ° C. for 45 minutes. 120 μl TE buffer (pH 7.5), then 1 μl 110 mg
Add / ml tRNA. Extract with 200 μl buffered phenol and re-extract with 100 μl TE buffer as described above. The two aqueous layers are combined and ethanol precipitated as described above.

C. Addition of linker to double-stranded cDNA At room temperature, in a 0.5 ml microcentrifuge tube: In order,
The total volume is 10 μl. After thoroughly mixing the contents by gentle pipetting, the tubes are briefly centrifuged in a microcentrifuge. Overnight at 16 ° C. or at room temperature (
(19-23 ° C) for 3-4 hours. Heat at 70 ° C. for 5 minutes to inactivate the ligase enzyme. This cDNA is usually digested with EcoRI to provide a sticky end of the cDNA for ligation into a vector and cleave the concatemer with a linker. The composition of the reaction solution is usually 10 μl of cDNA, 2 μl of 10 × EcoRI buffer (contents differ depending on the vendor), 2 μl of EcoRI (10 units / μl).
1 to sterile H ₂ O to make a total volume of 20 μl. The mixture is incubated at 37 ° C for 2-4 hours.

D. Size Fractionation of cDNA Usually, a linker molecule and a short (350 bp)
) Remove the cDNA fragment. For example, a plastic column (plastic 5)
can be used with a small piece of sterilized glass wool.
Prepare a ml Sepharose CL-4B column. The column is equilibrated with 1 × TE (10 mM Tris, 1 mM EDTA, pH 7.5) containing 0.1 M saline. Load the cDNA onto the column and collect 200 μl fractions. 2 of each fraction
The aliquots are analyzed by gel electrophoresis and autoradiography to determine the peak of the cDNA elution fraction. Usually, the fractions containing the first half of the peak are pooled, purified by ethanol precipitation and resuspended in 10 μl of distilled water.

E. Cloning of cDNA into Bacteriophage Vectors cDNAs designed for cloning and propagation of cDNA, previously digested with EcoRI and phosphorylated at the stump are commercially available (for example, Stratag).
ene company lambda gt10). The prepared cDNA is inserted into a lambda vector by ligation according to the manufacturer's manual. The ligated vector / cDNA molecule is packaged into phage particles using a commercially available packaging extract.

[0114]

Example 8 Screening of Human cDNA Library a. Labeling of DNA fragment (probe) for library screening An RNA probe that recognizes a nucleic acid sequence specific to NaN, such as NaN No. 1371-1751, is used. Other nucleotide sequences are nucleosides of human nucleic acid sequences.
It can be developed based on a NaN sequence such as Pseudo No. 765-1160 (FIGS. 2, 7 and 8). A HinDIII / BamHI fragment of NaN was inserted into a pBluescript (SK +) vector (Stratagene). Got
The sequence of the construct was confirmed by sequencing. The direction of the inserted DNA was HindII.
The 5 ′ and 3 ′ ends of the construct linearized at the restriction enzyme cleavage sites of I and BamHI, respectively, were placed at the T7 and T3 RNA polymerase promoters, respectively.
Closed. Digoxigenin-labeled sense (linearized with HindIII and transcribed with T7 RNA polymerase) and antisense (linearized with BamHI)
Transcript of T3 RNA polymerase) into MEGAscript transcription kit
(Ambion) and prepared in vitro based on the manufacturer's property table. Simple
Briefly, 1 μg of the linearized template was used for each RNA polymerase.
Depending on the enzyme, the following reagents are included:
1x enzyme mix containing RNase inhibitor and reaction buffer (Ambion
Co., 7.5 mM ATP, GTP and CTP nucleotides, 5.625 mM
 UTP and 1.725 mM Dig-11 UTP (Boehringer M
(Annheim) in a total volume of 20 μl. In vitro transcription reaction
Performed in a 37 ° C. water bath for 3 hours. Deoxyribonuclease free of ribonuclease
1 μl of Rease I (2 units / μl) is added to each reaction and added at 37 ° C. for another 15 minutes
The DNA template was removed by incubation. H without nuclease _Two O, 30 μl and LiCl solution (7.5 M lithium chloride, 50 mM EDTA
) 25 μl was added to stop the reaction.

The mixture was kept at −20 ° C. for 30 minutes. The RNA transcript was pelleted in a microfuge centrifuge at 13000 × g at 4 ° C. for 15 minutes. The supernatant was removed and the pellet was washed once with 100 μl of 75% ethanol. Mixture at room temperature 13000xg
And centrifuged again for 5 minutes. Thereafter, the pellet was air-dried in a closed chamber and subsequently dissolved in 100 μl of H ₂ O without ribonuclease. Transcript yield and integrity were determined using the DIG / Genius kit and the manufacturer (Boehring).
er Mannheim) and determined on agarose gel with formaldehyde in comparison with control DIG-labeled RNA. Alternatively, one skilled in the art can design radiolabeled probes for autoradiographic analysis.

Other regions of rat, mouse or human NaN sodium channel cDNA
For example, a 3 ′ untranslated region sequence or the like can be used as a probe for cDNA library screening or Northern blot analysis by the same method. In particular, the probe can be prepared using a commercially available kit such as an oligo labeling kit manufactured by Pharmacia and a Genius kit (Boehringer Mannheim).

B. cDNA library screening As described above, using a non-radioactive label (as described above) derived from the 3 'untranslated region of NaN or other regions or a NaN-specific probe of radiolabeled DNA or cRNA, using a moderately stringent Hybridization Wash (50 ~
(60 ° C., 5 × SSC, 30 minutes) to detect a recombinant plaque containing a full-length homolog of the human NaN sodium channel. Standard protocols involving the preparation of nitrocellulose or nylon filters loaded with recombinant phage (59,
Screen the library using 60). The recombinant DNA is then
Hybridize with an aN-specific probe (described above). Perform a moderately stringent wash.

Select plaques that are positive (ie, not artifacts or background) on multiple duplicate filters and perform subsequent purification. Usually, in order to separate phage, one or more screenings are performed after dilution. Thereafter, the resulting plaque is purified, the DNA is extracted, and the size of the cloned DNA is qualitatively analyzed. The clones are cross-hybridized with one another using standard techniques (59) to identify distinct positive clones.

[0119] Usually, duplicate clones encoding the channel are isolated. Next, using standard cloning techniques, any 5 ′ untranslated sequence, start codon ATG, coding sequence, stop codon and any 3 ′ untranslated sequence, poly A-added consensus sequence, and optionally poly A sequence, To produce a full-length cDNA construct. If the overlapping clone fragments do not generate enough fragments to constitute a full-length cDNA clone, alternative methods such as RACE (PCR-based) can be used to generate the missing or full-length clones.

C. Characterization of human-like full-length clones To compare the size of rat and mouse messenger RNA, the size of messenger RNA in human tissues was determined by Northern blot analysis using a NaN-specific cDNA sequence obtained from the full-length clone as a probe. I do.
Using a method similar to that described for rat NaN, cloned cDN
Confirm the biological activity of A.

[0121]

Example 9 Polymerase chain reaction using rat-derived DNA sequence (P
CR) Approach to cloning full length human NaN sodium channel by human dorsal root ganglia, trigeminal ganglia or other head and neck ganglia obtained from postmortem human, fetal or biopsy or surgically removed tissues with total RNA and polyA + RNA And isolated as described above. Then, as described in Example 1, a method for preparing cDNA and a PCR-based approach is used.

Degenerate PCR primers derived from rat NaN-specific coding sequence (see FIG. 2)
And amplify the cDNA by polymerase chain reaction (69). The skilled artisan can manipulate many variable operating conditions in a PCR reaction to obtain the required homologous sequence.

Such conditions include, but are not limited to, the temperature of each cycle and step, the number of cycles and steps, the thermotolerant polymerase, and the Mg ²⁺ concentration. Higher specificity can be achieved by using nested primers derived from more conserved sequences among the NaN sodium channels.

Typically, the amplification reaction is performed with 1 μl of first strand cDNA, 0.8 mM of each primer and E
xpand Long Template DNA polymerase mix (Bo
Performing in a total volume of 60 μl using 1.75 units (Ehringer Mannheim). The Expand Long Template DNA polymerase mix increases the yield of PCR products without increasing nonspecific amplification compared to conventional thermotolerant DNA polymerases and the like (49,50). The PCR reaction buffer is 50
It consists of mM Tris-HCl (pH 9.2), 16 mM (NH ₄ ) ₂ SO ₄ , 2.25 mM MgCl ₂ , 2% (V / V) DMSO and 0.1% Tween20. As described in (46) above, the amplification reaction is performed using a programmable thermal cycler (PTC).
-200, MJ Research, Cambridge, Mass.). First, a heat denaturation step was performed at 90 ° C. for 4 minutes, and an annealing step was performed for 6 minutes.
The extension reaction step is 90 minutes at 72 ° C. for 2 minutes at 0 ° C. Second, the heat denaturation step is at 94 ° C. for 1 minute, the annealing step is at 60 ° C. for 1 minute, and the extension reaction step is at 72 ° C. for 90 seconds. The second stage is 33 repeats with 35 cycles in all stages, extending the extension reaction step of the last cycle to 10 minutes. In addition, a control reaction is performed in parallel with the sample. This includes 1) c
Primers and all reaction components for all components excluding DNA (negative control), 2) constitutively expressed products (eg, GAPDH) should be used.

The products of the PCR reaction are examined on a 1-1.6% (W / V) agarose gel. A gel corresponding to the amplification reaction product of approximately the expected size (stained with edidium bromide,
(Observed by ultraviolet light)), and the DNA is purified.

The resulting DNA may be subjected to direct sequencing, for example, pCRII,
(Invitrogen) or pGEMT (Promega) and the like, but not limited thereto. Thereafter, the clones are sequenced to identify those containing sequences similar to the rat, mouse or human partial NaN sodium channel sequence.

[0127]

Example 10 Analysis of Clones The candidate clones obtained in Example 9 are further characterized by conventional techniques. Also, the biological activity of the expression product is confirmed by conventional techniques.

[0128]

Example 11 Isolation of Full Length NaN Sequence from Human Fetal Tissue A commercially available human fetal cDNA library and / or cDNA pool was prepared using the standard PCR protocol and standard screening procedure described above. Screen with primers or probes (for library screening).

[0129]

Example 12 Northern Blot of Rat DRG or Trigeminal Neurons with RatNaN Fragments Total RNA from whole DRG and / or DRG or trigeminal ganglia (as positively reactive tissues) and cerebral hemisphere, cerebellum and liver (as negatively reactive tissues) , 10
30 μg of denaturing 1% agarose-formaldehyde gel or agarose-
Electrophoresis in a glyoxal gel, then use standard molecular biology manuals (5
9, 60) and transferred to a nylon membrane as also described as being accomplished in multiple steps. A radiolabel (radiospecific activity of 10 ⁸ dpm / μg or more) or a digoxigenin-labeled RNA probe is usually used for Northern analysis. An antisense RNA probe (see Example 20, which describes in situ hybridization using a NaN-specific probe) was replaced with a sense DNA
It is created from fragments by in vitro synthesis. 6xSS membrane with immobilized RNA
C and place in a hybridization tube with the RNA side up. 1 ml prehybridization per 10 cm 2 of membrane
Add hybridization solution. Usually, prehybridization and hybridization are performed in a glass tube in a commercially available hybridization oven. The tube is placed in a hybridization oven and incubated at 60 ° C. for 15 minutes to 1 hour while rotating. Pipette the desired volume of probe into the hybridization tube and continue to incubate at 60 ° C. overnight while rotating. The probe concentration in the hybridization solution should be 10 ng / ml if the specific radioactivity is 10 ⁸ dpm / μg, or 2 ng / ml if the specific radioactivity is 10 ⁹ dpm / μg (digoxigenin labeling). Probes are used at 2-10 ng / ml).

Pour out the hybridization solution and add an equal volume of 2 × SSC / 0.1% SDS
Add. Incubate at room temperature for 5 minutes while rotating. Change wash fluid and repeat this step. To reduce the background, doubling the volume of the wash solution may also be useful. Wash solution with an equal volume of 0.2xSS
Replace with C / 0.1% SDS and incubate for 5 minutes at room temperature while rotating the tube.
Change wash solution and repeat this step (this is a low stringency wash). For moderate to highly stringent conditions, additional washing is performed with a wash liquor preheated to a moderate temperature (42 ° C.) or high temperature (68 ° C.). Remove the final wash and rinse the membrane in 2 × SSC at room temperature. Autoradiography is then performed for up to one week.
Alternatively, if a non-radioactive probe is used, the chemiluminescent technology (Amers
ham company). The signal from the gel is compared with a standard molecular weight marker for gel and the size of the transcript is calculated.

[0131]

Example 13 Tissue-Specific Distribution of NaN by RT-PCR Method NaN-specific forward (5′CCCTGCTGCGCTCGGTGAAGAA3
RT) using cDNA prepared from a plurality of rats as a template, using primers') and reverse (5 'GACAAAGTAGATCCCAGAGG3') as primers.
PCR was performed. These primers amplify between nucleotides 765 and 1156 (392 bp) in the NaN sequence, due to the absence of similar sequences in the database (using a program such as the National Library of Medicine BLASTN). Judgment is NaN specific. Usually, the amplification reaction is performed using the first strand cDNA 1
μl, 0.8 μM of each primer and Expand Long Template
Using 1.75 units of DNA polymerase enzyme mix (Boehringer Mannheim), the reaction was carried out in a total volume of 60 μl. The Expand Long Template DNA polymerase enzyme mix can be used with conventional thermotolerant DN.
Increases the yield of PCR products without increasing non-specific amplification compared to A polymerase and the like (49, 50). The PCR reaction buffer was 50 mM Tris-HCl (p
H9.2), 16 mM (NH ₄ ) ₂ SO ₄ , 2.25 mM MgCl ₂ , 2% (V / V
) Consists of DMSO and 0.1% Tween20. As described in (71) above, the amplification reaction was performed using a programmable thermal cycler (PTC-200, MJRese).
arch, Cambridge, Mass.) in two steps. First
In addition, a heat denaturation step was performed at 94 ° C. for 4 minutes, followed by an annealing step at 60 ° C. for 2 minutes and an extension reaction step at 72 ° C. for 90 seconds. Second, the heat denaturation step was at 94 ° C. for 1 minute, the annealing step was at 60 ° C. for 1 minute, and the extension reaction step was at 72 ° C. for 90 seconds. The second stage consisted of 33 repeats, 25 to 35 cycles in all stages, and the extension reaction step in the final cycle was extended to 10 minutes.

Glyceraldehyde triphosphate dehydrogenase (GAPDH) was used as an internal control to confirm that the lack of NaN signal in different tissues was not due to template degradation or PCR inhibitors. The rat GAPDH sequence was co-amplified using primers that amplify a 66 bp product corresponding to nucleotides 328-994 (Accession number: M17701). Based on the structure of the human gene, amplification products span multiple exon-intron junctions (72)
. Prior to the reverse transcription reaction, treatment with deoxyribonuclease I was carried out as usual in order to prevent amplification of the GAPDH sequence from a pseudogene expression product of the genome that might contaminate the adjusted total RNA.

It is primarily and preferentially expressed in NaNDRG and trigeminal ganglion neurons. FIG. 4 shows RT-PCR performed on various neural and non-neural tissues.
3 shows the results of the screening by. Lanes 1, 2, 4, 9 and 16 are 400
The single amplification product that migrated with the bp marker is consistent with the 392 bp NaN-specific product. Lanes 1 and 16, 2, 4 and 9 are D
Includes products using RG, cerebral hemisphere, retina and trigeminal ganglion. According to this assay, NaN was expressed in the cerebellum, optic nerve, spinal cord, sciatic nerve, frontal cranial ganglion (superior cr
anial ganglia), skeletal muscle, myocardium, adrenal gland, uterus, liver or kidney (lane 3 respectively)
, 5-8, and 10-15). PC which went in parallel
In the R reaction, the amplification product of GAPDH was also obtained (data not shown), so that the signal of NaN was attenuated in the cerebral hemisphere and retina and was not detected in other tissues. Alternatively, it can be seen that it is not due to the inhibitor of PCR in the cDNA template.

[0134]

Example 14 Transformation of host cells with NaN coding sequences Transformed host cells for the measurement of sodium current or intracellular sodium ion levels are usually constructed from cells such as HEK293 cells (constructs).
At the same time, it is prepared by simultaneously transducing a fluorescent reporter plasmid (pGreen Lantern-1, Life Technologies) using the calcium phosphate precipitation method (27). HEK293 cells usually contain 10
% Sucrose DME supplemented with 5% fetal calf serum (Life Technologies)
Culture in D medium (Life Technologies). 48 hours later,
Green fluorescent cells are selected for recording (28).

To prepare a cell line that constitutively expresses a recombinant channel, the NaN construct is cloned into a vector comprising a selectable marker for other mammalian cells. The calcium phosphate precipitation method is used for transduction (27). Human fetal kidney cells (
HEK-293), Chinese hamster ovary cells (CHO) and other suitable cell lines are cultured under standard tissue culture conditions in Dulbecco's Modified Eagle's Medium supplemented with 10% fetal calf serum. Add a DNA / calcium phosphate mixture to the cell culture,
After 15-20 hours, the cells are washed with fresh medium. 48 hours later, antibiotics (G
418, Geneticin, Life Technologies) to select cells that have acquired the neomycin resistance trait. After 2-3 weeks at G418,
Harvest 10-20 isolated colonies using the tip of a sterile 10 ml pipette. Colonies are cultured for an additional 4-7 days, split and subsequently tested for channel expression by whole-cell patch-clamp recording and RT-PCR.

[0136]

Example 15 Production of NaN-Specific Antibodies Using antigenic NaN-specific peptides, rabbits are raised to polyclonal antibodies to produce antibodies specific to rat, mouse or human NaN. As an example, the peptide chain CGPNPASNK meets the criteria of immunogenicity and surface accessibility.
D C FEKEKDSED (rat amino acid No. No. 285-304) is selected.
This peptide sequence does not match any sequence in public databases. The underlined cysteine (C) residue is alanine (C) to prevent disulfide bond formation.
A). It is expected that this amino acid change will not cause a great difference in antibody specificity.

Peptide synthesis, rabbit immunization, and affinity purification of anti-peptide antibodies were by standard techniques. The purified antibodies were tested against DRG in culture. Using these antibodies, immunostaining was performed according to standard procedures before and after blocking with excess peptide.

After culturing the DRG neurons for 16 to 24 hours, they were treated as described below, and the NaN protein was detected immunochemically. Physiological saline (137 mM NaCl, 5.3 mM KC
L, 1 ITIMM902 25 mM sorbitol, 10 mM HEPES, 3 m
Wash the coverslip with M CaCl ₂ pH 7.2), fix in 4% paraformaldehyde / 0.14 M phosphate buffer at 4 ° C. for 10 minutes, and wash 3 times with phosphate buffered saline (PBS) for 5 minutes. After washing, the cells were blocked for 15 minutes with PBS containing 20% normal goat serum, 1% bovine serum albumin, and 0.1% Triton X-100. These cover glasses were incubated overnight at 4 ° C. in an anti-NaN antibody (100-fold diluted solution). After overnight incubation, the cover glass was thoroughly washed with PBS, and then incubated with Cy3-labeled anti-rabbit IgG (1: 3000, Amersham) at room temperature for 2 hours. After the cover glass was washed with PBS, it was mounted on a slide glass with Aqua Polymount. The neurons were examined with a LightAristoplan light microscope equipped with a fluorescence wavelength unit, using a Dage DC330T color camera and a Sci.
Images were recorded with an onCG-7 color PCI frame capture device (see FIG. 7).

[0139]

Example 16 In the neuropathic pain model, the expression of NaN fluctuates. The plasticity of sodium channel expression in DRG neurons was examined using the Neuropathic Pain CCI model (Bennett and Xie). Twenty-two adult Sprague-Dawley female rats weighing 240-260 g were anesthetized with pentobarbital sodium salt (50 mg / kg iv), and the right sciatic nerve was exposed at the center of the thigh. The method of Bennette and Xie (1988,
Pain 33, 87-107) and loosely ligated 4 needles around the nerve with chrome gut surgical thread (No. 4-0). The incision was layered and closed, and bacteriostatic antibiotics were intramuscularly injected.

Already, sciatic nerve transection shows dramatic changes in sodium currents in axotomized DRGs, in parallel with the transcripts of various sodium channels in these neurons. It is known. The TTX-R sodium current and the two TTX-R sodium channels (SNS / PN3 and NaN) were significantly attenuated, while the rapidly repriming TT
XS currents become apparent and transcripts of the αIII sodium channel, which produce TTX-S currents, are up-regulated. The present inventors have found that 14 days after surgery, in contrast to axotomy, DRG undergoes CCI-induced changes. Like TTX-R sodium current, TTX-specific for two sensory neurons
The transcripts of the R channels NaN and SNS are significantly down-regulated, while the transcript of the TTX-S αIII sodium channel has a small diameter D
Upregulated in RG neurons. These changes can be part of the hyperexcitability of DRG neurons that contribute to hyperalgesia caused by trauma.

[0141]

Example 17 Assay for Drugs that Modulate NaN Channel Activity Using Patch Clamping Using cells that express cloning Na + channels, drugs that modulate NaN channel activity, eg, block or inhibit channels Alternatively, assay for agents that promote channel opening. Because channel activity is voltage-dependent, depolarizing conditions may be used to observe baseline activity modulated by the agent being tested. Depolarization can be accomplished by any available means, including, for example, increasing the extracellular potassium ion concentration to about 20-40 nM or repeatedly applying an electrical pulse.

The agent to be tested is cultured with HEK293 cells or other transformed cells expressing a Na + channel (28). After incubation for a sufficient period of time, drug-induced changes in Na + channel activity can be measured by patch-clamping (29). The data for these measurements was obtained on a Macintosh Quadra 950 or similar computer using Pulse (version 7.52, HEK, Germany).
(Company A). Glass capillaries from Sutter P-
Using a 87 puller machine or similar device, smooth the tip with a flame and use the electrode (0.8
(1.5 MW). Usually, for cells, the initial value of the sealing voltage is less than 5 G ohm, and the leakage current is large (the holding current at -80 m is less than 0.1 nA).
The analysis is only considered if there are bubbles in the membrane and the access resistance is less than 5 Mohm. Monitor the access resistance value and do not use the data if the resistance value fluctuates. A series compensation resistor is used to minimize voltage errors and, if necessary, cancel out capacitance drift with a computer controlled amplifier or similar technique.

In order to enable comparison between voltage-dependent activation and inactivation, cells are usually used whose maximum voltage fluctuation after compensation is 10 mV or less. A linear subtraction method is used to take a voltage clamp record. The membrane current is typically filtered at 5 KHz and sampled at 20 KHz. Pipette _{solution, 140mM CsF, 2mM MgCl 2,} 1mM EGTA and 10 mM Na-HEPES
(PH 7.3). Standard paging solutions were 140 mM NaCl, 3 mM KCl, 2 mM MgCl ₂ , 1 mM CaCl ₂ , 10 mM H
Standard solutions such as EPES, and 10 mM glucose (pH 7.3).

Pre-pulses that are exposed to appropriate concentrations of TTX and / or distinguish between trototoxin (TTX) resistance and TTX-sensitive Na + currents based on their different steady-state (potential) inactivation properties Measure TTX resistance and TTX sensitive current by protocol.

Data is collected and analyzed using standard pulse protocols to determine sodium current properties such as potential dependence, steady state (potential) state properties, kinetics, and repriming. By measuring the amount of current and the capacitance of the cells, it is possible to estimate the Na + current density and to compare the channel densities under different conditions based on this (22, 30). By examining cells under current clamp conditions, the pattern of spontaneous and evoked action potential generation, threshold of occurrence, response frequency characteristics,
In addition, depolarization / hyperpolarization and other aspects of power generation are examined (55). These measurements are made on both control cells expressing NaN and cells exposed to the agent being tested.

[0146]

Example 18 Intracellular sodium [Na⁺Assay for Drugs that Modulate NaN Channel Activity by Measurement The drug to be tested is cultured with cells that exhibit NaN channel activity. sufficient
After a long period of culture, the drug-induced Na⁺The change in channel activity was determined using SBFI and the relative intensity of the image [Na⁺] Measure the amount. In this method, a Na such as SBFI (sodium-binding benzofuran isophthalate (33)) is used. ⁺ Indicator or a similar dye, and thus free [Na⁺] Measure
I do. First, the acetoxymethyl ester of SFBI, which is membrane permeable to cells (
SBFI / AM) or a similar dye (usually dimethyl sulfoxide (DMSO
) Is dissolved to a concentration of 10 mM to prepare a stock solution).
A commercially available relative light intensity measuring device (for example, Georgia Instruments)
Signal intensity on a microscope stage using a microscope. The appropriate wavelength (
For example, excitation light of 340: 385 nm) is supplied. The excitation light is converted to a circular dichroic reflector (
(400 nm) to pass through the cells and collect the emission of 450 nm or more. firefly
The optical signal is, for example, an image intensifier (such as GenIISyS).
Amplified by a CCD camera or similar device.
Interfaced to the bar. In consideration of the decay of the fluorescence, free in the cytoplasm [Na ⁺ ] Uses a wavelength ratio of 340: 385.

To calibrate the fluorescence intensity of SBFI, cells are perfused with a calibration solution containing a known concentration of Na ⁺ (typically 0 and 30 mM, or 0, 30 and 50 mM).
M [Na +], and gramicidin and monensin). According to Rose and Ransom (34), the 345/390 fluorescence ratio of SBFI varies monotonically with changes in [Na ⁺ ] i concentration. Experiments were repeated with multiple (typically at least 4) different coverslips, and statistics were obtained on control cells and cells exposed to various concentrations of drugs that block, inhibit, or enhance channel activity. To obtain highly significant measurement results. Such a method is Sonthei
described by Mer et al. (32).

[0148]

Embodiment 19 NaN channel by scintigraphic imaging
Assay for Drugs that Modulate Cell Activity⁺Agents that modulate NaN channel activity, such as blocking, inhibiting, or inhibiting channels, using cell lines that express channels
Assay for agents that promote the opening of the. For example, if the drug to be tested is Na⁺Culture with HEK293 expressing the channel, or other transformed cells (28). Ten
After culturing for a short period of time, Na isotope method⁺Was measured and the Na induced by the drug was measured.⁺Detect changes in channel activity.^{twenty two}Na is a gamma ray emitter and Na⁺(31), and can be used to measure the flow rate of³⁶Rb⁺Is Na Channel
(32). ⁸⁶ Rb⁺Ion is K⁺It is taken in by Na + / K + ATPase like ion,
Its half-life is⁴²K⁺It has the advantage of being much longer than (35). Therefore, one-way ouabain sensitivity³⁶Rb⁺By measuring the uptake of Na, the activity of Na + / K + ATPase, which is the source of the action potential, can be quantitatively assayed.
Wear.

After culturing NaN-expressing cells and labeling them with isotopes, the intracellular isotope content is measured by a liquid scintillation counter or a similar method, and the amount of cellular proteins is determined by the bicinchoninic acid protein according to the modified method for cultured cells (37). Determined by assay (36) or the like. The flow rates of ²² Na and ³⁶ Rb ⁺ are determined in the presence or absence of agents that block and inhibit Na + or promote channel opening. This makes it possible to determine the effect of these drugs on NaN.

[0150]

Example 20 In Situ Hybridization a. Probes Probes are prepared as described in Example 5.

B. Culture of DRG neurons Culture of DRG neurons from adult rats has been established and has been reported (70). Briefly, the supporting tissues and myelin of the lumbar ganglia (L4, L5) from adult Sprague-Dawley female rats are removed, and cultured in an enzyme solution containing collagenase and then papain. Tissues were cultured in 1: 1 Dulbecco's Modified Eagle's Medium (DMEM) and Hanks' F12 medium, 10% fetal bovine serum, 1.5 mg / ml trypsin inhibitor, 1.5 mg / ml bovine serum albumin, 100 units / ml penicillin, and The cells were triturated in a culture solution containing 0.1 mg / ml streptomycin, and the cells were seeded at a density of 500 to 1000 cells / mm ² on a cover glass coated with polyornithine / laminin. The cells are maintained at 37 ° C. overnight in a humidified 95% air / 5% CO ² incubator and then processed for in situ hybridization and cytochemistry as previously described (56,57). Those skilled in the art can also culture trigeminal ganglia in a similar manner.

C. Tissue Preparation Mature Sprague-Dawley female rats are deeply anesthetized with, for example, chloral hydrate, and first with a phosphate buffered saline (PBS) solution, and then with sorensen phosphate buffer containing 4% paraformaldehyde, pH 7.4. Was perfused through the heart at 4 ° C. After perfusion fixation, the dorsal root ganglia and trigeminal ganglia at L4 and L5 sites were harvested and placed in fresh fixative. After 2-4 hours, the tissues were placed in 0.14M phosphate buffer containing 4% paraformaldehyde and 30% Sucrose and stored at 4 ° C overnight. 15 μm tissue sections were cut and placed on glass slides coated with poly-L-lysine. Slides are processed for in situ hybridization and cytochemistry as previously reported (24,56). After in situ hybridization and cytochemical treatment, slides were dehydrated, cleaned, and mounted with Permount. FIG. 5 shows the result.

DRG sections hybridized with the NaN sense riboprobe showed no non-specific labeling (FIG. 5, panel C). In the DRG hybridized with the NaN antisense riboprobe (FIG. 5, panel A) and the trigeminal ganglion (panel B), the signal of NaN is present in most small neurons (<30 mm in diameter). Most of the large neurons (30 mm or more in diameter) did not show signals due to NaN hybridization. No specific signal was observed in sections of notochord, cerebellum and liver hybridized with the antisense NaN riboprobe (panels D, E and F, respectively).

[0154]

Example 21 Microsatellite Sequence The mouse intron microsatellite sequence is shown below. These microsatellites could be used as markers to screen for mutant alleles associated with the disease that may show polymorphisms in the human SCN11a gene. Hybridization or amplification assays are readily available as such screening methods. Sambrook
See, e.g., K. et al.

Intron 4: microsatellite is dTdg AGTTTAATGTTGAGTGAATTGTGGTGGTGATTTCCCACTTGAGGCCTTTGTGTTAAAGCCCAATGT
GTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGGTTGGGGGGTGGTGGCAGAGT
CTGGTATTGGTAAGGTGAGAGCAATCCCAGAACGTCCACCTGCTCTTCCATTTTATTAATCAGGCAGGCCTC
T intron 5: micro satellite is dCdTdG (dNdG2) _x (X5-30) GTAAGCCACTGGCTCTTAACTAAAATGCTCGTTGGCATTAGAACATTTCTGAGCTGGGGTGGTGGT
GGTGGTGGTGGTGGTGGTGGTGGTGGTGGTGGTGGTGGTGGTGGTGATGGTGGTGGTGGAGGTGGNGGTGGA
GGTGGTGGCTGTGGTGGTGGNGGTGGTGGTGGTGGTGGANGTGGANGTGGTGGCGTGGTGGTGGNGGTGGTG
GTGGAGGTGGTGGCTGTGGTGGTNGTGGTGGC Intron 6: microsatellite is dCdA TGTGCATGCTTGATTCCCAGCTCCTATGGTCTGATTACTCGGTCCTTAGGAGCAAGGCCAGACTGT
CCACCCTGACACACACACACACACACACACACACACACACACACACACACACACACAGTGTAGAGAATTACC
TCATTCTTGGAGTTTCTCTGGAAAAGGAATGTCTCAAAGCCAAGTTCACAGAGCAACAGCTG Intron 8: 5 'microsatellite stretches dTdCg followed by dT TGTTAGAAACTCTAAGACAATGAAGCACCATGCTGGAAATAAGAGCACAAACTCTTTCTTCATGCA
TTACCCACTGCTTGTGCTTTCACCTTAGTGCTCGTGCTCTCTCTTTCTCTCTCTCTCTCTCTCTCTCTCTCTCT
CTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTTTTTTTTTTTTTTTT Intron 8: 3 'microsatellite is dCdA CACACACACACACACACACACACACACACACACACACACAGAGAAACACTGTCGCAGTCATACATA
TAAAGATAAATACATCTTAAAAAAAGAACCATGTGATTGAGTTATAAAATATTCCAACTTAT Intron 10B: microsatellite dCdA, followed by dCdA ₃ nucleotides downstream dCdA ₃ AGGTCATTTCCTCTGCAGTGTGCTTGGCAGGAAAAACTTCCTGGCTATTCAAGTCAGTGCCCTGCT
TGATCATCCATGTATCACACACACACAAAACAAACAAACAAACAAACAAAACCCTGGGGAAGAAGGAAGAGG
TTAAGCACATAGGCAGAGAGCAGCCAGGCTGACTCAGAGCAAACACCTGATCATTCTTCCAT Intron 12: Microsatellite dpydG (dt / dCdG) GTGCTGGGATCAAAGGCGTGCGCCGCCACCACGCCCGGCCCCTTTTTATGTTTCAAATTTACTTTT
ATCATGTGCACGTGTGTGGGTGCGTGCATGTGTGTGCGTGCGTGTGCGTGTGNGTGTGNGTGTGTGTGTGTGTG
TGTGTGTGTGTGTGTGTGTG Intron 14: microsatellite is dCdA CACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACTTGCATCTT
TGAGTTAATTGGATAGGCTGAGTCTTACACCGGAATCATACTGTTGC Intron 15A: micro satellite is dCdA CCAATGAGAGACTCTTGTCTCAAAAAAGCCATGGTGTCCAGATCCTGAGGAATAACACCTAAGAAT
GTGCTCTGGCCTGAAAACACACACACACACACACACACACACACACACACACACAGTTTTATTTATTTATTT
AAAAAAATATGTCTCTAGGCATTGCTGAAATGTCTCCTACAGGATTAAGTCAACCAGAGCCA It should be understood that the above discussion and examples merely present a detailed description of certain preferred embodiments. It will be apparent to those skilled in the art that various modifications and equivalent changes can be made without departing from the spirit and scope of the invention.

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[Brief description of the drawings]

FIG. 1 shows the sequence of rat NaN cDNA.

FIG. 2 shows the putative amino acid sequence of rat NaN cDNA. The predicted cell membrane portion of domains I-IV is underlined. DI- associated with the TTX-R phenotype
The amino acid of SS2, serine "S", is shown in bold type.

FIG. 3 shows a schematic diagram of the predicted secondary structure of the α-subunit of NaN.

FIG. 4 shows the results of RT-PCR analysis of α-NaN of extracts from various tissues using NaN-specific primers. NaN is abundantly expressed in spinal and trigeminal ganglia. Low levels of NaN are detected in the cerebral hemisphere and retinal tissue. No NaN signal is detected in the cerebellum, optic nerve, spinal cord, osteotomy, upper cervical ganglion, skeletal muscle, myocardium, adrenal gland, uterus, liver and kidney.

FIG. 5 shows tissue distribution of α-NaN by in situ hybridization. All tissues are from grown spray dolly rats. Scale bar = 50 μm

FIG. 5A. Trigeminal ganglion cells show moderate to high hybridization signals.

FIG. 5B. Dorsal root ganglion cells show moderate to high hybridization signals in their small cells. In large dorsal root ganglion cells, the hybridization signal is attenuated (arrow).

FIG. 5C. In DRG neurons, there is no sense probe signal.

FIG. 5D. No hybridization signal is seen in the spinal cord.

FIG. 5E. No hybridization signal is seen in the cerebellum.

FIG. 5F. No hybridization signal is seen in the liver.

FIG. 6 shows the predicted length of the amplified product of domain 1 of the rat α-subunit and the restriction enzyme profile specific for that subunit.

FIG. 7A shows nucleotides of mouse NaN.

FIG. 7B shows the amino acid sequence of mouse NaN.

FIG. 8A shows a partial nucleotide sequence of the human NaN protein.

FIG. 8B shows a partial amino acid sequence of the human NaN protein.

FIG. 9: Obtained from adult rat L4 / 5 ganglia, reacting with antibodies to NaN
1 shows a culture of DRG neurons processed for immunofluorescence localization.

FIG. 9ab. NaN immunostaining is prominent in the cell bodies of DRG neurons.

FIG. 9c. NaN is present in the cell body as well as in neurites of DRG neurons.

FIG. 9d is a Normalsky photograph of a neuron that does not express NaN protein.

FIG. 9d ′ is a fluorescence photograph of a neuron that does not express NaN protein.

FIG. 10 shows the location of Scn11a and its related genes on the 9-terminal of the mouse chromosome.

FIG. 10A: Haptotype from Jackson BSS backcross. Black box is C57BL / 6
J represents aryl, open box represents SPRET / Ei aryl. The number of animals with each haptotype is shown below each row. When typing was ambiguous, missing data was inferred from nearby data.

FIG. 10B is a map of the chromosome 9 terminal based on the data of FIG. 10A. The positions of Scn5a and Scn10a from the MGD consensus map and the human ortholog position are marked. The number is the cM position on the consensus map (http: //www.infor
matics.jax.org/bin/ccr/index).

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) A61P 43/00 111 C07K 16/18 4C085 C07K 16/18 C12P 21/02 C 4H045 C12N 5/10 G01N 33 / 566 15/09 ZNA 33/84 Z C12P 21/02 A61K 37/02 G01N 33/566 C12N 5/00 B 33/84 15/00 ZNAA (81) Designated countries EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), AU, CA, JP, US F term (reference) 2G045 AA40 BB20 BB24 CB01 CB17 CB26 CB30 DA12 DA13 DA14 DA36 DA80 DB09 FA16 FA19 FA20 FA34 FB02 FB03 FB05 FB06 FB07 FB08 FB12 GC19 GC20 4B024 AA01 AA11 BA80 CA04 DA01 DA02 DA05 DA11 EA04 GA11 4B064 AG01 CA19 C C24 DA13 4B065 AA01X AA57X AA87X AA93Y AB01 AC14 BA02 CA24 CA46 4C084 AA02 AA17 BA03 CA53 CA62 DC50 ZA052 ZA082 ZB112 4C085 AA13 AA14 BB11 CC05 DD22 DD33 EE01 4H045 AA40 FA10 BA10

Claims (29)

[Claims] 1. A nucleic acid molecule comprising the sequence shown in FIG. 1, 7A, or 8A, and a nucleic acid molecule encoding the sequence of FIG. 2, 7B, 8B, or an allelic variant of the sequence. An isolated nucleic acid molecule selected from the group consisting of: a nucleic acid molecule that hybridizes under stringent conditions to one of the above sequences. 2. The isolated nucleic acid molecule of claim 1, wherein said nucleic acid encodes a voltage-gated Na ⁺ channel that is selectively expressed in the dorsal root ganglion or trigeminal ganglion. 3. The isolated nucleic acid molecule of claim 2, wherein said nucleic acid encodes a human NaN sodium channel. An expression vector comprising the isolated nucleic acid according to any one of claims 1 to 3 alone or together with appropriate regulatory and expression control elements. A host cell transformed with the expression vector according to claim 4. 6. A Na ⁺ channel encoded by the isolated nucleic acid according to any one of claims 1 to 3. 7. The Na ⁺ channel according to claim 6, having the amino acid sequence of FIG. 8. A protein molecule having the amino acid sequence of FIG. 2, FIG. 7B or FIG. 8B, or the amino acid sequence of a peptide fragment thereof. 9. A protein encoded by the isolated nucleic acid according to claim 6. 10. A method for identifying an agent that modulates the activity of a Na ⁺ channel according to claim 6, comprising the step of contacting the agent with a cell that expresses the Na ⁺ channel on its surface. The resulting change in sodium current, change in membrane potential,
Or a step of measuring a change in intracellular Na ⁺ . 11. The method according to claim 10, wherein the measuring step is achieved by a voltage clamp measurement or a measurement of the membrane potential. 12. The method according to claim 10, wherein said measuring step is achieved by measuring the concentration of intracellular sodium. 13. The method according to claim 10, wherein said measuring step is accomplished by measuring sodium influx. 14. The method of claim 13, wherein said influx of sodium is measured using ²² Na or ⁸⁶ Rb. 15. A method for identifying an agent that regulates the transcription or translation of mRNA encoding the Na ⁺ channel according to claim 6, wherein the agent is a cell that expresses the Na ⁺ channel on its surface. Contacting and measuring the resulting level of Na ⁺ channel expression. 16. Administering an effective amount of an agent capable of altering the flow of Na ⁺ current through NaN channels in DGD or trigeminal neurons to produce pain, A method of treating paresthesia and / or hypersensitivity symptoms. 17. Pain, paresthesia or the like in an animal or human subject by administering an effective amount of an agent capable of modulating the transcription or translation of the mRNA encoding the Na ⁺ channel of claim 6. And / or a method of treating hypersensitivity symptoms. 18. An isolated nucleic acid that is antisense to the nucleic acid of claim 1 and is long enough to regulate expression of a NaN channel in a cell containing mRNA. 19. Administering a labeled monoclonal antibody or other labeled ligand specific to the NaNNa ⁺ channel to image the locus of onset of pain or to produce a DGD or A scintigraphic method that provides a measure of the level of pain associated with hypersensitivity symptoms mediated by trigeminal neurons. 20. A method for identifying a tissue, a cell and a cell type that expresses a NaN sodium channel, comprising a step of detecting NaN on a cell surface or in a cell. 21. A method for identifying a NaN-expressing tissue, cell and cell type, comprising detecting mRNA encoding NaN therein. 22. A method for producing a transformed cell that expresses an exogenous nucleic acid encoding NaN, wherein the isolated nucleic acid according to any one of claims 1 to 3 comprises Transforming said cells with an expression vector comprising regulatory and expression control elements. 23. An isolated antibody specific for a NaN channel or a polypeptide fragment thereof. 24. The method according to claim 23, wherein the antibody is labeled.
The antibody according to 1. 25. A set comprising a step of culturing the transformed host according to claim 5 under conditions in which a NaN sodium channel or protein is expressed, and a step of recovering the NaN protein. A method for producing a recombinant NaN protein. 26. Alter the flow of rapidly repriming currents in axotomized, inflamed, or otherwise damaged DRG neurons (eg, by increasing or decreasing). A) a therapeutic composition comprising an effective amount of a drug capable of 27. The method according to claim 26, comprising administering the therapeutic composition according to claim 26.
A method of treating acute pain, acute or chronic, nervous or inflammatory pain and hypersensitivity symptoms in an animal or human subject. 28. Testing for the ability to up-regulate or down-regulate NaN channel mRNA in axotomized, inflamed, or otherwise damaged DRG neurons. And screening for candidate compounds for use in treating hypersensitivity symptoms. 29. NaN allele in a nucleic acid sample comprising a step of hybridizing the intron microsatellite sequence of Example 20 to a nucleic acid under conditions of sufficient stringency to generate a distinct signal. A method for screening gene variants.