JP2000201684A - Sodium channel scn8 - Google Patents
Sodium channel scn8Info
- Publication number
- JP2000201684A JP2000201684A JP11004645A JP464599A JP2000201684A JP 2000201684 A JP2000201684 A JP 2000201684A JP 11004645 A JP11004645 A JP 11004645A JP 464599 A JP464599 A JP 464599A JP 2000201684 A JP2000201684 A JP 2000201684A
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Abstract
Description
【０００１】[0001]
【発明の属する技術分野】この出願は、新規なナトリウ
ムチャンネル分子ＳＣＮ８Ａと、この分子のｃＤＮＡに
関するものである。さらに詳しくは、この出願は、ヒト
神経系のナトリウムチャンネルの新規なαサブユニット
ＳＣＮ８Ａと、そのｃＤＮＡに関するものである。The present application relates to a novel sodium channel molecule SCN8A and the cDNA of this molecule. More specifically, this application relates to a novel α subunit SCN8A of the sodium channel of the human nervous system and its cDNA.
【０００２】[0002]
【従来の技術】膜電位依存性ナトリウムチャンネルは、
神経・筋などの興奮性細胞の細胞膜に存在する膜タンパ
ク質分子である。興奮性細胞は、活動電位を発生させる
ことによってその機能を果たしている。例えば、神経細
胞の場合には、細胞体において発生した活動電位が軸索
を伝播することによって、神経細胞の機能である情報の
伝達が行われる。ナトリウムチャンネルは、カリウムチ
ャンネルとともに、活動電位の開始・伝播を担っている
タンパク質分子である。BACKGROUND OF THE INVENTION Membrane voltage-gated sodium channels are
It is a membrane protein molecule present in the cell membrane of excitable cells such as nerves and muscles. Excitable cells perform their function by generating action potentials. For example, in the case of a nerve cell, information, which is a function of the nerve cell, is transmitted by the action potential generated in the cell body propagating through the axon. Sodium channels are protein molecules that are responsible for the initiation and propagation of action potentials together with potassium channels.
【０００３】膜依存性ナトリウムチャンネルは複数のサ
ブユニットから構成されているが、その主要な機能はα
サブユニットに備わっている。これまでに、哺乳動物に
おいては複数のαサブユニット遺伝子の存在が明らかに
されており、ヒトにおいては少なくともＳＣＮ１Ａ（2q
24）、ＳＣＮ２Ａ（2q23）、ＳＣＮ３Ａ（2q24-q31）、
ＳＣＮ４Ａ（17q23.1-q25.3）、ＳＣＮ５Ａ（3p21）、
ＳＣＮ６Ａ（2q21-q23）、ＳＣＮ８Ａ（12q13）、ＳＣ
Ｎ９Ａ、ＳＣＮ１０Ａの９個の遺伝子が明らかにされて
いる（括弧内はヒト染色体座）。ただし、これら全ての
遺伝子が完全な形でクローニングされている訳ではな
く、遺伝子によっては部分的な配列のみが報告されてい
るものや、あるいはマウスやラットの相同配列からヒト
相同遺伝子が確認されているものも存在する。特に、Ｓ
ＣＮ８Ａは、完全長のｃＤＮＡは未だに特定されておら
ず、その遺伝子がコードするタンパク質についても全貌
は知られていない。[0003] The membrane-gated sodium channel is composed of several subunits, the main function of which is α
Provided in subunit. To date, the existence of multiple α subunit genes has been elucidated in mammals, and at least SCN1A (2q
24), SCN2A (2q23), SCN3A (2q24-q31),
SCN4A (17q23.1-q25.3), SCN5A (3p21),
SCN6A (2q21-q23), SCN8A (12q13), SC
Nine genes of N9A and SCN10A have been identified (the human chromosomal loci in parentheses). However, not all of these genes have been cloned in their complete form.Some of them have only been reported as partial sequences, or human homologous genes have been identified from mouse or rat homologous sequences. Some exist. In particular, S
The full-length cDNA of CN8A has not yet been identified, and the entire protein encoded by that gene is not known.
【０００４】近年、ヒトまたは動物においてこれら遺伝
子の突然変異による疾患が同定されており、ナトリウム
チャンネルのαサブユニットは、生理学的に重要な分子
というだけでなく、その変異が疾患原因となることも明
らかにされてきている。例えば、ＳＣＮ４Ａの突然変異
により家族性高カリウム血症性周期性四肢麻痺が生じる
こと、ＳＣＮ５Ａの突然変異によりＱＴ延長症候群１型
が発症することなどが明らかにされている。また、ヒト
での相応する疾患は知られてはいないが、マウスミュー
タント motor endplate disease (med)の原因遺伝子と
してＳｃｎ８ａがクローニングされている。In recent years, diseases caused by mutations in these genes have been identified in humans or animals. The α subunit of the sodium channel is not only a physiologically important molecule, but the mutation may cause disease. It has been revealed. For example, it has been revealed that mutation of SCN4A causes familial hyperkalemia periodic limb paralysis, and mutation of SCN5A causes long QT syndrome type 1. Although no corresponding disease in humans is known, Scn8a has been cloned as a causative gene of mouse mutant motor endplate disease (med).
【０００５】[0005]
【発明が解決しようとする課題】前記のとおり、膜依存
性ナトリウムチャンネルの分子生物学的実体と機能を解
明することは、ヒトの生理メカニズムを理解するうえで
も、また、各種疾患の直接原因を特定するうえでも極め
て重要である。さらには、その生理的機能から推察し
て、ナトリウムチャンネルに作用する物質はそれが発現
している細胞の機能を調節する薬物（例えば、麻酔薬、
鎮痛薬、筋弛緩剤など）となる可能性があり、新たな治
療薬剤開発の標的としても重要である。As described above, elucidation of the molecular biological entity and function of a membrane-gated sodium channel is necessary for understanding the physiological mechanism of humans and for determining the direct cause of various diseases. It is extremely important for identification. Furthermore, inferring from its physiological functions, substances acting on sodium channels are drugs that regulate the function of cells in which they are expressed (eg, anesthetics,
Analgesics, muscle relaxants, etc.) and are also important targets for the development of new therapeutic agents.
【０００６】この出願の発明者らは、ヒト神経系で発現
している全てのナトリウムチャンネルを確認することを
目的として遺伝子のクローニングを行った結果、ヒトの
ＳＣＮ８Ａの完全長ｃＤＮＡをクローニングし、このc
ＤＮＡがコードしているＳＣＮ８Ａの全アミノ酸配列を
明らかにすることに成功した。この出願は、発明者らが
見いだしたヒトのナトリウムチャンネルＳＣＮ８Ａと、
そのｃＤＮＡを具体的に提供することを課題としてい
る。またこの出願は、ＳＣＮ８Ａの部分配列からなるペ
プチド、ｃＤＮＡの部分配列からなるＤＮＡ断片、並び
にＳＣＮ８Ａに対する抗体を提供することを課題として
いる。The inventors of the present application cloned a gene for the purpose of confirming all sodium channels expressed in the human nervous system, and as a result, cloned a human SCN8A full-length cDNA. c
We succeeded in elucidating the entire amino acid sequence of SCN8A encoded by the DNA. This application describes a human sodium channel SCN8A that the inventors have discovered,
It is an object to provide the cDNA specifically. Another object of the present application is to provide a peptide consisting of a partial sequence of SCN8A, a DNA fragment consisting of a partial sequence of cDNA, and an antibody against SCN8A.
【０００７】[0007]
【課題を解決するための手段】この出願は、前記の課題
を解決する発明として、配列番号１のアミノ酸配列を有
するナトリウムチャンネルＳＣＮ８Ａを提供する。ま
た、この出願は、前記のナトリウムチャンネルＳＣＮ11
Ａをコードするヒト遺伝子を提供する。The present invention provides a sodium channel SCN8A having the amino acid sequence of SEQ ID NO: 1 as an invention for solving the above-mentioned problems. This application also relates to the aforementioned sodium channel SCN11.
A human gene encoding A is provided.
【０００８】さらにこの出願は、上記ヒト遺伝子のｃＤ
ＮＡであって、配列番号１の塩基配列を有するｃＤＮ
Ａ、および配列番号２の塩基配列を有するｃＤＮＡを提
供する。この出願はまた、前記のナトリウムチャンネル
ＳＣＡ11Ａに対する抗体をも提供する。[0008] Further, the present application relates to the cD of the human gene.
NA, a cDN having the base sequence of SEQ ID NO: 1
A and a cDNA having the nucleotide sequence of SEQ ID NO: 2. This application also provides antibodies to the sodium channel SCA11A described above.
【０００９】以下、発明の実施形態を示し、この出願の
発明についてさらに詳しく説明する。Hereinafter, embodiments of the invention will be described, and the invention of this application will be described in more detail.
【００１０】[0010]
【発明の実施の形態】この発明のナトリウムチャンネル
ＳＣＮ８Ａは、配列番号１に塩基配列を示したｃＤＮＡ
から発現される転写産物であり、配列番号１に示した19
80個のアミノ酸からなっている。この発明のＳＣＮ８Ａ
は、公知の方法、すなわちヒトの各種臓器から単離する
方法、この出願によって提供されるのアミノ酸配列に基
づき化学合成によってペプチドを調製する方法、あるい
はこの出願によって提供されるｃＤＮＡを用いて組換え
ＤＮＡ技術で生産する方法などにより取得することがで
きる。例えば、組換えＤＮＡ技術によってＳＣＮ８Ａを
取得する場合には、この発明のｃＤＮＡを有するベクタ
ーからインビトロ転写によってＲＮＡを調製し、これを
鋳型としてインビトロ翻訳を行なうことにより、ＳＣＮ
８Ａタンパク質を得ることができる。またｃＤＮＡの翻
訳領域を公知の方法により適当な発現ベクターに組換
え、この組換えベクターで大腸菌、枯草菌、酵母、動植
物細胞等を形質転換すれば、これらの形質転換体でＳＣ
Ｎ８Ａを大量に発現させることができる。BEST MODE FOR CARRYING OUT THE INVENTION The sodium channel SCN8A of the present invention is a cDNA having the nucleotide sequence shown in SEQ ID NO: 1.
And a transcript expressed from
Consists of 80 amino acids. SCN8A of the invention
Are known methods, that is, a method of isolation from various human organs, a method of preparing a peptide by chemical synthesis based on the amino acid sequence provided by this application, or a recombinant method using cDNA provided by this application. It can be obtained by a method using DNA technology. For example, when SCN8A is obtained by recombinant DNA technology, RNA is prepared from a vector having the cDNA of the present invention by in vitro transcription, and this is used as a template for in vitro translation to obtain SCN8A.
8A protein can be obtained. Moreover, if the cDNA translation region is recombined into an appropriate expression vector by a known method, and this recombinant vector is used to transform Escherichia coli, Bacillus subtilis, yeast, animal and plant cells, etc., these transformants will
N8A can be expressed in large amounts.
【００１１】この発明のＳＣＮ８Ａをインビトロ翻訳で
生産する場合には、この発明のｃＤＮＡの翻訳領域をＲ
ＮＡポリメラーゼプロモーターを有するベクターに組換
え、プロモーターに対応するＲＮＡポリメラーゼを含む
ウサギ網状赤血球溶解物や小麦胚芽抽出物などのインビ
トロ翻訳系に添加すればよい。ＲＮＡポリメラーゼプロ
モーターとしては、Ｔ７、Ｔ３、ＳＰ６などが例示でき
る。これらのＲＮＡポリメラーゼプロモーターを含むベ
クターとしては、ｐＫＡ１、ｐＣＤＭ８、ｐＴ３／Ｔ７
１８、ｐＴ７／３ １９、ｐＢluescript IIなどが例
示できる。When the SCN8A of the present invention is produced by in vitro translation, the translation region of the cDNA of the present invention is converted to R
It may be recombined into a vector having an NA polymerase promoter and added to an in vitro translation system such as a rabbit reticulocyte lysate or wheat germ extract containing an RNA polymerase corresponding to the promoter. Examples of the RNA polymerase promoter include T7, T3, SP6 and the like. Vectors containing these RNA polymerase promoters include pKA1, pCDM8, pT3 / T7
18, pT7 / 319, pBluescript II and the like.
【００１２】また、この発明のＳＣＮ８Ａを大腸菌など
の微生物で発現させる場合には、微生物中で複製可能な
オリジン、プロモーター、リボソーム結合部位、ｃＤＮ
Ａクローニング部位、ターミネーター等を有する発現ベ
クターに、この発明のｃＤＮＡの翻訳領域を組換えて発
現ベクターを作成し、この発現ベクターで宿主細胞を形
質転換し、この形質転換体を培養すればよい。この際、
任意の翻訳領域の前後に開始コドンと停止コドンを付加
すれば、任意の領域を含むタンパク質断片を得ることが
できる。あるいは、他のタンパク質との融合タンパク質
として発現させることもできる。この融合タンパク質を
適当なプロテアーゼで切断することによってＳＣＮ８Ａ
のみを取得することもできる。大腸菌用発現ベクターと
しては、ｐＵＣ系、ｐＢluescript II、ｐＥＴ発現シス
テム、ｐＧＥＸ発現システムなどが例示できる。When the SCN8A of the present invention is expressed in a microorganism such as Escherichia coli, the origin, promoter, ribosome binding site, cDN
The translation region of the cDNA of the present invention may be recombined into an expression vector having an A cloning site, a terminator and the like to prepare an expression vector, a host cell may be transformed with the expression vector, and the transformant may be cultured. On this occasion,
By adding a start codon and a stop codon before and after an arbitrary translation region, a protein fragment containing the arbitrary region can be obtained. Alternatively, it can be expressed as a fusion protein with another protein. Cleavage of this fusion protein with an appropriate protease allows SCN8A
You can also get only. Examples of expression vectors for Escherichia coli include pUC system, pBluescript II, pET expression system, pGEX expression system, and the like.
【００１３】この発明のＳＣＮ８Ａを真核細胞で発現さ
せる場合には、この発明のｃＤＮＡの翻訳領域を、プロ
モーター、スプライシング領域、ポリ（Ａ）付加部位等
を有する真核細胞用発現ベクターに組換え、真核細胞内
に導入する。発現ベクターとしては、ｐＫＡ１、ｐＣＤ
Ｍ８、ｐＳＶＫ３、ｐＭＳＧ、ｐＳＶＬ、ｐＢＫ−ＣＭ
Ｖ、ｐＢＫ−ＲＳＶ、ＥＢＶベクター、ｐＲＳ、ｐＹＥ
Ｓ２などが例示できる。真核細胞としては、サル腎臓細
胞ＣＯＳ７、チャイニーズハムスター卵巣細胞ＣＨＯな
どの哺乳動物培養細胞、出芽酵母、分裂酵母、カイコ細
胞、アフリカツメガエル卵細胞などが一般に用いられる
が、これらに限定されるものではない。発現ベクターを
真核細胞に導入するには、電気穿孔法、リン酸カルシウ
ム法、リポソーム法、ＤＥＡＥデキストラン法など公知
の方法を用いることができる。When the SCN8A of the present invention is expressed in eukaryotic cells, the translation region of the cDNA of the present invention is recombined into an expression vector for eukaryotic cells having a promoter, a splicing region, a poly (A) addition site and the like. , Into eukaryotic cells. Examples of expression vectors include pKA1, pCD
M8, pSVK3, pMSG, pSVL, pBK-CM
V, pBK-RSV, EBV vector, pRS, pYE
S2 and the like can be exemplified. As eukaryotic cells, mammalian cultured cells such as monkey kidney cells COS7, Chinese hamster ovary cells CHO, budding yeast, fission yeast, silkworm cells, Xenopus egg cells and the like are generally used, but are not limited thereto. . In order to introduce the expression vector into eukaryotic cells, known methods such as an electroporation method, a calcium phosphate method, a liposome method, and a DEAE dextran method can be used.
【００１４】上記の方法により原核細胞や真核細胞でタ
ンパク質を発現させたのち、培養物から目的タンパク質
を単離精製するためには、公知の分離操作を組み合わせ
て行う。例えば、尿素などの変性剤や界面活性剤による
処理、超音波処理、酵素消化、塩析や溶媒沈殿法、透
析、遠心分離、限外濾過、ゲル濾過、ＳＤＳ−ＰＡＧ
Ｅ、等電点電気泳動、イオン交換クロマトグラフィー、
疎水性クロマトグラフィー、アフィニティークロマトグ
ラフィー、逆相クロマトグラフィー等である。After the protein is expressed in prokaryotic cells or eukaryotic cells by the above-described method, a known separation operation is combined to isolate and purify the target protein from the culture. For example, treatment with a denaturant such as urea or a surfactant, ultrasonic treatment, enzymatic digestion, salting out or solvent precipitation, dialysis, centrifugation, ultrafiltration, gel filtration, SDS-PAG
E, isoelectric focusing, ion exchange chromatography,
Hydrophobic chromatography, affinity chromatography, reverse phase chromatography and the like.
【００１５】この発明のＳＣＮ８Ａには、配列番号２の
アミノ酸配列におけるいかなる部分配列を含むペプチド
断片（５アミノ酸残基以上）も含まれる。これらのペプ
チド断片は抗体を作製するための抗原として用いること
ができる。また、この発明のＳＣＮ1Ａには、他の任意
のタンパク質との融合蛋白質も含まれる。この発明の遺
伝子は、上記ＳＣＮ８Ａをコードするヒトの遺伝子であ
って、ヒト染色体１２番長腕（12q13）に存在する遺伝
子である。この遺伝子は、例えばこの発明のｃＤＮＡま
たはその一部配列をプロ−ブとして、既存のゲノムライ
ブラリ−から単離することができる。The SCN8A of the present invention includes a peptide fragment (5 amino acid residues or more) containing any partial sequence in the amino acid sequence of SEQ ID NO: 2. These peptide fragments can be used as antigens for producing antibodies. The SCN1A of the present invention also includes a fusion protein with any other protein. The gene of the present invention is a human gene encoding the above-mentioned SCN8A, which is present on the long arm of human chromosome 12 (12q13). This gene can be isolated from an existing genomic library, for example, using the cDNA of the present invention or a partial sequence thereof as a probe.
【００１６】この発明のｃＤＮＡは、例えばヒト細胞由
来のｃＤＮＡからクローン化することができる。ｃＤＮ
Ａはヒト細胞から抽出したポリＡ+ＲＮＡを鋳型として
合成する。合成法法としては、岡山−Berg法（Mol. Cel
l. Biol. 2:161-170, 1982）、Gubler−Hoffman 法(J.
Gene 25:263-269, 1983）、キャッピング法（Gene 150:
243-250,1994）等を用いることができる。また市販のヒ
トｃＤＮＡライブラリーを用いることもできる。ｃＤＮ
Ａライブラリーからこの発明のｃＤＮＡをクローン化す
るには、この発明のｃＤＮＡの任意の部分の塩基配列に
基づいてオリゴヌクレオチドを合成し、これをプローブ
として用いて、公知の方法によりコロニーあるいはプラ
ークハイブリダイゼーションによるスクリーニングを行
えばよい。また、目的とするｃＤＮＡ断片の両末端にハ
イブリダイズするオリゴヌクレオチドを合成し、これを
プライマーとして用いて、ヒト細胞から単離したｍＲＮ
ＡからＲＴ−ＰＣＲ法により、この発明のｃＤＮＡを調
製することもできる。The cDNA of the present invention can be cloned, for example, from cDNA derived from human cells. cDN
A synthesizes using poly A + RNA extracted from human cells as a template. Okayama-Berg method (Mol. Cel)
l. Biol. 2: 161-170, 1982), Gubler-Hoffman method (J.
Gene 25: 263-269, 1983), capping method (Gene 150:
243-250, 1994). A commercially available human cDNA library can also be used. cDN
To clone the cDNA of the present invention from the A library, an oligonucleotide is synthesized based on the nucleotide sequence of an arbitrary portion of the cDNA of the present invention, and this is used as a probe to colony or plaque hybrid by a known method. Screening by hybridization may be performed. In addition, an oligonucleotide that hybridizes to both ends of the cDNA fragment of interest is synthesized, and using this as a primer, mRN isolated from human cells is used.
The cDNA of the present invention can also be prepared from A by the RT-PCR method.
【００１７】なお、一般にヒト遺伝子は個体差による多
型が頻繁に認められる。従って配列番号１または２にお
いて、１または複数個のヌクレオチドの付加、欠失およ
び／または他のヌクレオチドによる置換がなされている
ｃＤＮＡもこの発明のｃＤＮＡに含まれる。同様に、こ
れらの塩基の変更によって生じる１または複数個のアミ
ノ酸の付加、欠失および／または他のアミノ酸による置
換がなされているＳＣＮ８Ａも、配列番号１または２の
アミノ酸配列を有するＳＣＮ８Ａの活性を有する限り、
この発明に含まれる。Generally, polymorphism due to individual differences is frequently observed in human genes. Accordingly, the cDNA of SEQ ID NO: 1 or 2 in which one or more nucleotides have been added, deleted and / or substituted with other nucleotides are also included in the cDNA of the present invention. Similarly, SCN8A in which one or more amino acids are added, deleted, and / or substituted by another amino acid resulting from the change of these bases also has the activity of SCN8A having the amino acid sequence of SEQ ID NO: 1 or 2. As long as you have
Included in this invention.
【００１８】この発明のｃＤＮＡには、配列番号１また
は２の塩基配列のいかなる部分配列を含むＤＮＡ断片
（10bp以上）も含まれる。また、センス鎖およびアンチ
センス鎖からなるＤＮＡ断片も含まれる。これらのＤＮ
Ａ断片は遺伝子診断用のプローブ等として用いることが
できる。この発明の抗体は、ＳＣＮ８Ａそれ自体、また
はその部分ペプチドを抗原として、公知の方法によりポ
リクロ−ナル抗体またはモノクロ−ナル抗体として得る
ことができる。The cDNA of the present invention includes a DNA fragment (10 bp or more) containing any partial sequence of the nucleotide sequence of SEQ ID NO: 1 or 2. It also includes a DNA fragment consisting of a sense strand and an antisense strand. These DNs
The A fragment can be used as a probe for gene diagnosis and the like. The antibody of the present invention can be obtained as a polyclonal antibody or a monoclonal antibody by a known method using SCN8A itself or a partial peptide thereof as an antigen.
【００１９】次に実施例として、この発明のＳＣＮ８Ａ
の取得経緯およびその特性等についての検討結果を示
す。Next, as an embodiment, the SCN8A of the present invention will be described.
The following shows the details of the acquisition process and the results of its characteristics.
【００２０】[0020]
【実施例】（１）ヒト剖検脳ｍＲＮＡの精製 非神経疾患患者剖検脳片２ｇを20mLのTRIzol試薬（Gibc
o-BRL社製）と共にホモジェナイザーにて完全に破砕
し、30℃、15分間インキュベートした。次に、４mLのク
ロロフォルムを加え、攪拌した後、30℃、30分間インキ
ュベートした。12,000ｇ、15分間遠心し、水層を分取
し、エタノール沈殿によりtotalＲＮＡを抽出した。ダ
イナビーズｍＲＮＡ精製キット（DYNAL社製）を用いてt
otalＲＮＡよりポリ+ＲＮＡ（ｍＲＮＡ）分画を精製し
た。 （２）ＲＴ−ＰＣＲ スーパークリプトII逆転写酵素（Gibco-BRL社製）によ
り、（１）で精製したｍＲＮＡを鋳型として、ランダム
プライマーを用いて１本鎖ＤＮＡを合成した。この１本
鎖ＤＮＡを鋳型とし、図１に示したように既知のナトリ
ウムチャンネルαサブユニット（ヒトＨおよびラット
Ｒ）間において相同性が高く、配列のよく保存されてい
る部分を基に設計したオリゴヌクレオチドをプライマー
とするネストＰＣＲにより、既知のナトリウムチャンネ
ルαサブユニットを含むナトリウムチャンネルの部分配
列を得た。そのうちの一つ1806bpのクローンはラットで
クローニングされているＳｃｎ８ａと対応する部分の相
同性が塩基配列91％、アミノ酸配列で98％と非常に高い
ことから、このクローンがヒトＳＣＮ８ＡｃＤＮＡの部
分配列であることが確認された。 （３）ＲＡＣＥ 上記（２）で得たヒトＳＣＮ８ＡｃＤＮＡの部分配列の
塩基配列を基にプライマーを合成し、Marathon-ready c
DNA（Clontech社製）を鋳型として５’−ＲＡＣＥおよ
び３’−ＲＡＣＥを行い、５’側および３’側のｃＤＮ
Ａを得た。また、５’側の末端部分はラットの５’側の
末端ｃＤＮＡ配列をもとに設計したプライマーを用いて
ＰＣＲ法により得た。得られた各ｃＤＮＡ断片の関係は
図２に示したとおりである。 （４）サブクローニング ＲＴ−ＰＣＲおよびＲＡＣＥによって得たＳＣＮ８Ａの
ｃＤＮＡ断片をプラスミドベクターpBleuscript IISK
(+)にサブクローニングした。 （５）シークエンシング 上記（４）のプラスミドを増殖精製し、ダイターミネー
ターおよびダイプライマー法によりシークエンシング反
応を行い、反応産物を蛍光シークエンサー３７７Ａ（Pe
rkin Elmer/ABI）にて解析した。 （６）完全長ｃＤＮＡ発現クローンの作製 上記（１）で精製したｍＲＮＡを鋳型として、ＲＴ−Ｐ
ＣＲにより全長のＯＲＦを含むＳＣＮ８ＡのｃＤＮＡを
得、これらをプラスミドベクターｐＳＰ６４ＴＲ（Prom
ega社製のｐＳＰ６４Ｐoly(A)を変形したもの）にサブ
クローニングした（図３）。 （７）結果と考察 ＳＣＮ８ＡのｃＤＮＡは、配列番号１に示したとおり、
全長7053bpで、5940のＯＲＦを有し、1980アミノ酸残基
のタンパク質をコードしている。このＳＣＮ８Ａには、
ナトリウムチャンネルαサブユニットに共通する４カ所
の膜貫通ドメインが同定される。EXAMPLES (1) Purification of human autopsy brain mRNA 2 g of autopsy brain slice from a patient with non-neurological disease was added to 20 mL of TRIzol reagent (Gibc
o-BRL) with a homogenizer and incubated at 30 ° C. for 15 minutes. Next, 4 mL of chloroform was added, stirred, and then incubated at 30 ° C. for 30 minutes. The mixture was centrifuged at 12,000 g for 15 minutes, the aqueous layer was separated, and total RNA was extracted by ethanol precipitation. T using Dynabeads mRNA purification kit (DYNAL)
The poly + RNA (mRNA) fraction was purified from otal RNA. (2) RT-PCR A single-stranded DNA was synthesized by supercrypt II reverse transcriptase (manufactured by Gibco-BRL) using the mRNA purified in (1) as a template and random primers. Using this single-stranded DNA as a template, as shown in FIG. 1, the known sodium channel α subunits (human H and rat R) were designed based on a highly homologous and well conserved part of the sequence. A partial sequence of a sodium channel containing a known sodium channel α subunit was obtained by nest PCR using an oligonucleotide as a primer. One of the clones, 1806 bp, has a very high homology of 91% in base sequence and 98% in amino acid sequence corresponding to Scn8a cloned in rat, and thus this clone is a partial sequence of human SCN8A cDNA. It was confirmed that. (3) RACE Primers were synthesized based on the base sequence of the partial sequence of human SCN8A cDNA obtained in (2) above, and Marathon-ready c
5′-RACE and 3′-RACE were performed using DNA (manufactured by Clontech) as a template, and 5′- and 3′-side cDN
A was obtained. The 5'-terminal portion was obtained by PCR using primers designed based on the rat 5'-terminal cDNA sequence. The relationship between the obtained cDNA fragments is as shown in FIG. (4) Subcloning The cDNA fragment of SCN8A obtained by RT-PCR and RACE was transformed into a plasmid vector pBleuscript IISK.
Subcloned into (+). (5) Sequencing The plasmid of (4) above was propagated and purified, and subjected to a sequencing reaction by a dye terminator and a dye primer method, and the reaction product was subjected to a fluorescent sequencer 377A (Pe
rkin Elmer / ABI). (6) Preparation of full-length cDNA expression clone Using the mRNA purified in the above (1) as a template, RT-P
The SCN8A cDNA containing the full-length ORF was obtained by CR, and these were used as plasmid vectors pSP64TR (Prom
(a modified version of pSP64Poly (A) manufactured by ega) (FIG. 3). (7) Results and Discussion The cDNA of SCN8A was, as shown in SEQ ID NO: 1,
It has a total length of 7053 bp, an ORF of 5940, and encodes a protein of 1980 amino acid residues. In this SCN8A,
Four transmembrane domains common to the sodium channel α subunit are identified.
【００２１】表１には、既知のナトリウムチャンネルα
サブユニット遺伝子ｃＤＮＡとの相同性を示した。マウ
スおよびラットの相同遺伝子との相同性はそれぞれ86％
および94％であった。また、既知のαサブユニットのう
ちでは、ＳＣＮ１Ａ、ＳＣＮ２Ａ、ＳＣＮ３Ａとの間に
比較的高い相同性が認められた。Table 1 shows the known sodium channel α
It showed homology with the subunit gene cDNA. 86% homology with mouse and rat homologous genes respectively
And 94%. In addition, among the known α subunits, relatively high homology was observed with SCN1A, SCN2A, and SCN3A.
【００２２】[0022]
【表１】 [Table 1]
【００２３】[0023]
【発明の効果】以上詳しく説明したとおり、この出願に
よって、ヒトのナトリウムチャンネルαサブユニットＳ
ＣＮ８ＡとそのｃＤＮＡが提供される。これらの発明
は、興奮性細胞の関与する生理メカニズムの解明、各種
のヒト疾患の原因の特定、および新たな治療薬剤の開発
に大きく貢献する。As described in detail above, according to this application, human sodium channel α subunit S
CN8A and its cDNA are provided. These inventions will greatly contribute to elucidation of the physiological mechanism involving excitable cells, identification of the causes of various human diseases, and development of new therapeutic agents.
【００２４】[0024]
【配列表】 SEQUENCE LISTING <110> 科学技術振興事業団 <120> ナトリウムチャンネルＳＣＮ８Ａ <160> 1 <170> PatentIn Ver. 2.0 <210> 1 <211> 7053 <212> DNA <213> Homo sapiens <220> <221> CDS <222> (1)..(5940) <400> 1 atg gcg gcg cgg gtg ctt gca cca cca ggc cct gat agt ttc aag cct 48 Met Ala Ala Arg Val Leu Ala Pro Pro Gly Pro Asp Ser Phe Lys Pro 1 5 10 15 ttc acc cct gag tca ctg gca aac att gag agg cgc att gct gag agc 96 Phe Thr Pro Glu Ser Leu Ala Asn Ile Glu Arg Arg Ile Ala Glu Ser 20 25 30 aag ctc aag aaa cca cca aag gcc gat ggc agt cat cgg gag gac gat 144 Lys Leu Lys Lys Pro Pro Lys Ala Asp Gly Ser His Arg Glu Asp Asp 35 40 45 gag gac agc aag ccc aag cca aac agc gac ctg gaa gca ggg aag agt 192 Glu Asp Ser Lys Pro Lys Pro Asn Ser Asp Leu Glu Ala Gly Lys Ser 50 55 60 ttg cct ttc atc tac ggg gac atc ccc caa ggc ctg gtt gca gtt ccc 240 Leu Pro Phe Ile Tyr Gly Asp Ile Pro Gln Gly Leu Val Ala Val Pro 65 70 75 80 ctg gag gac ttt gac cca tac tat ttg acg cag aaa acc ttt gta gta 288 Leu Glu Asp Phe Asp Pro Tyr Tyr Leu Thr Gln Lys Thr Phe Val Val 85 90 95 tta aac aga ggg aaa act ctc ttc aga ttt agt gcc acg cct gcc ttg 336 Leu Asn Arg Gly Lys Thr Leu Phe Arg Phe Ser Ala Thr Pro Ala Leu 100 105 110 tac att tta agt cct ttt aac ctg ata aga aga ata gct att aaa att 384 Tyr Ile Leu Ser Pro Phe Asn Leu Ile Arg Arg Ile Ala Ile Lys Ile 115 120 125 ttg ata cat tca gta ttt agc atg atc att atg tgc act att ttg acc 432 Leu Ile His Ser Val Phe Ser Met Ile Ile Met Cys Thr Ile Leu Thr 130 135 140 aac tgt gta ttc atg act ttt agt aac cct cct gac tgg tcg aag aat 480 Asn Cys Val Phe Met Thr Phe Ser Asn Pro Pro Asp Trp Ser Lys Asn 145 150 155 160 gtg gag tac acg ttc aca ggg att tat aca ttt gaa tca cta gtg aaa 528 Val Glu Tyr Thr Phe Thr Gly Ile Tyr Thr Phe Glu Ser Leu Val Lys 165 170 175 atc att gca aga ggt ttc tgc ata gat ggc ttt acc ttt tta cgg gat 576 Ile Ile Ala Arg Gly Phe Cys Ile Asp Gly Phe Thr Phe Leu Arg Asp 180 185 190 cca tgg aac tgg tta gat ttc agt gtc atc atg atg gcg tat ata aca 624 Pro Trp Asn Trp Leu Asp Phe Ser Val Ile Met Met Ala Tyr Ile Thr 195 200 205 gag ttt gta aac cta ggc aat gtt tca gct cta cgc act ttc agg gta 672 Glu Phe Val Asn Leu Gly Asn Val Ser Ala Leu Arg Thr Phe Arg Val 210 215 220 ctg agg gct ttg aaa act att tcg gta atc cca ggc ctg aag aca att 720 Leu Arg Ala Leu Lys Thr Ile Ser Val Ile Pro Gly Leu Lys Thr Ile 225 230 235 240 gtg ggt gcc ctg att cag tct gtg aag aaa ctg tca gat gtg atg atc 768 Val Gly Ala Leu Ile Gln Ser Val Lys Lys Leu Ser Asp Val Met Ile 245 250 255 atg aca gtg ttc tgc ctg agt gtt ttt gcc ttg atc gga ctg cag ctg 816 Met Thr Val Phe Cys Leu Ser Val Phe Ala Leu Ile Gly Leu Gln Leu 260 265 270 ttt cat ggg gaa cct tcg aac aag tgt gtt gtg tgg ccc ata aac ttc 864 Phe His Gly Glu Pro Ser Asn Lys Cys Val Val Trp Pro Ile Asn Phe 275 280 285 aac gag agc tat ctt gaa aat ggc acc aaa ggc ttt gat tgg gaa gag 912 Asn Glu Ser Tyr Leu Glu Asn Gly Thr Lys Gly Phe Asp Trp Glu Glu 290 295 300 tat atc aac aat aaa aca aat ttc tac aca gtt cct ggc atg ctg gaa 960 Tyr Ile Asn Asn Lys Thr Asn Phe Tyr Thr Val Pro Gly Met Leu Glu 305 310 315 320 cct tta ctc tgt ggg aac agt tct gat gct ggg caa tgc cca gag gga 1008 Pro Leu Leu Cys Gly Asn Ser Ser Asp Ala Gly Gln Cys Pro Glu Gly 325 330 335 tac cag tgt atg aaa gca gga agg aac ccc aac tat ggt tac aca agt 1056 Tyr Gln Cys Met Lys Ala Gly Arg Asn Pro Asn Tyr Gly Tyr Thr Ser 340 345 350 ttt gac act ttt agc tgg gcc ttc ttg gca tta ttt cgc ctt atg acc 1104 Phe Asp Thr Phe Ser Trp Ala Phe Leu Ala Leu Phe Arg Leu Met Thr 355 360 365 cag gac tat tgg gaa aac ttg tat caa ttg act tta cga gca gcc ggg 1152 Gln Asp Tyr Trp Glu Asn Leu Tyr Gln Leu Thr Leu Arg Ala Ala Gly 370 375 380 aaa aca tac atg atc ttc ttc gtc ttg gtc atc ttt gtg ggt tct ttc 1200 Lys Thr Tyr Met Ile Phe Phe Val Leu Val Ile Phe Val Gly Ser Phe 385 390 395 400 tat ctg gtg aac ttg atc ttg gct gtg gtg gcc atg gct tat gaa gaa 1248 Tyr Leu Val Asn Leu Ile Leu Ala Val Val Ala Met Ala Tyr Glu Glu 405 410 415 cag aat cag gca aca ctg gag gag gca gaa caa aaa gag gct gaa ttt 1296 Gln Asn Gln Ala Thr Leu Glu Glu Ala Glu Gln Lys Glu Ala Glu Phe 420 425 430 aaa gca atg ttg gag caa ctt aag aag caa cag gaa gag gca cag gct 1344 Lys Ala Met Leu Glu Gln Leu Lys Lys Gln Gln Glu Glu Ala Gln Ala 435 440 445 gct gcg atg gcc aat tca gca gga act gtc tca gaa gat gcc ata gag 1392 Ala Ala Met Ala Asn Ser Ala Gly Thr Val Ser Glu Asp Ala Ile Glu 450 455 460 gaa gaa ggt gaa gaa gga ggg ggc tcc cct cgg agc ttt tct gaa atc 1440 Glu Glu Gly Glu Glu Gly Gly Gly Ser Pro Arg Ser Phe Ser Glu Ile 465 470 475 480 tct aaa atc agc tca aag agt gca aag gaa aga agt aac agg aga aag 1488 Ser Lys Ile Ser Ser Lys Ser Ala Lys Glu Arg Ser Asn Arg Arg Lys 485 490 495 aag agg aag caa aag gaa ctc ttt gaa gga gag gag aaa ggg gat ccc 1536 Lys Arg Lys Gln Lys Glu Leu Phe Glu Gly Glu Glu Lys Gly Asp Pro 500 505 510 gag aag gtg ttt aag tca gag tca gaa gat ggc atg aga agg aag gcc 1584 Glu Lys Val Phe Lys Ser Glu Ser Glu Asp Gly Met Arg Arg Lys Ala 515 520 525 ttt cgg ctg cca gac aac aga ata ggg agg aaa ttt tcc atc atg aat 1632 Phe Arg Leu Pro Asp Asn Arg Ile Gly Arg Lys Phe Ser Ile Met Asn 530 535 540 cag tca atg ttc agc atc cca ggc tcg ccc ttc ctc tcc cgc cac aac 1680 Gln Ser Met Phe Ser Ile Pro Gly Ser Pro Phe Leu Ser Arg His Asn 545 550 555 560 agc aag agc agc atc ttc agt ttc agg gga cct ggg cgg ttc cga gac 1728 Ser Lys Ser Ser Ile Phe Ser Phe Arg Gly Pro Gly Arg Phe Arg Asp 565 570 575 ccg ggc tcc gag aat gag ttc gcg gat gac gag cac agc acg gtg gag 1776 Pro Gly Ser Glu Asn Glu Phe Ala Asp Asp Glu His Ser Thr Val Glu 580 585 590 gag agc gag ggc cgc cgg gac tcc ctc ttc atc ccc atc cgg gcc cgc 1824 Glu Ser Glu Gly Arg Arg Asp Ser Leu Phe Ile Pro Ile Arg Ala Arg 595 600 605 gag cgc cgg agc agc tac agc ggc tac agc ggc tac agc cag ggc agc 1872 Glu Arg Arg Ser Ser Tyr Ser Gly Tyr Ser Gly Tyr Ser Gln Gly Ser 610 615 620 cgc tcc tcg cgc atc ttc ccc agc ctg cgg cgc agc gtg aag cgc aac 1920 Arg Ser Ser Arg Ile Phe Pro Ser Leu Arg Arg Ser Val Lys Arg Asn 625 630 635 640 agc acg gtg gac tgc aac ggc gtg gtg tcc ctc atc ggc ggc ccc ggc 1968 Ser Thr Val Asp Cys Asn Gly Val Val Ser Leu Ile Gly Gly Pro Gly 645 650 655 tcc cac atc ggc ggg cgt ctc ctg cca gag gct aca act gag gtg gaa 2016 Ser His Ile Gly Gly Arg Leu Leu Pro Glu Ala Thr Thr Glu Val Glu 660 665 670 att aag aag aaa ggc cct gga tct ctt tta gtt tcc atg gac caa tta 2064 Ile Lys Lys Lys Gly Pro Gly Ser Leu Leu Val Ser Met Asp Gln Leu 675 680 685 gcc tcc tac ggg cgg aag gac aga atc aac agt ata atg agt gtt gtt 2112 Ala Ser Tyr Gly Arg Lys Asp Arg Ile Asn Ser Ile Met Ser Val Val 690 695 700 aca aat aca cta gta gaa gaa ctg gaa gag tct cag aga aag tgc ccg 2160 Thr Asn Thr Leu Val Glu Glu Leu Glu Glu Ser Gln Arg Lys Cys Pro 705 710 715 720 cca tgc tgg tat aaa ttt gcc aac act ttc ctc atc tgg gag tgc cac 2208 Pro Cys Trp Tyr Lys Phe Ala Asn Thr Phe Leu Ile Trp Glu Cys His 725 730 735 ccc tac tgg ata aaa ctg aaa gag att gtg aac ttg ata gtt atg gac 2256 Pro Tyr Trp Ile Lys Leu Lys Glu Ile Val Asn Leu Ile Val Met Asp 740 745 750 cct ttt gtg gat tta gcc atc acc atc tgc atc gtc ctg aat aca ctg 2304 Pro Phe Val Asp Leu Ala Ile Thr Ile Cys Ile Val Leu Asn Thr Leu 755 760 765 ttt atg gca atg gag cac cat cct atg aca cca caa ttt gaa cat gtc 2352 Phe Met Ala Met Glu His His Pro Met Thr Pro Gln Phe Glu His Val 770 775 780 ttg gct gta gga aat ctg gtt ttc act gga att ttc aca gcg gaa atg 2400 Leu Ala Val Gly Asn Leu Val Phe Thr Gly Ile Phe Thr Ala Glu Met 785 790 795 800 ttc ctg aag ctc ata gcc atg gat ccc tac tat tat ttc caa gaa ggt 2448 Phe Leu Lys Leu Ile Ala Met Asp Pro Tyr Tyr Tyr Phe Gln Glu Gly 805 810 815 tgg aac att ttt gac gga ttt att gtc tcc ctc agt tta atg gaa ctg 2496 Trp Asn Ile Phe Asp Gly Phe Ile Val Ser Leu Ser Leu Met Glu Leu 820 825 830 agt cta gca gac gtg gag ggg ctt tca gtg ctg cga tct ttc cga ttg 2544 Ser Leu Ala Asp Val Glu Gly Leu Ser Val Leu Arg Ser Phe Arg Leu 835 840 845 ctc cga gtc ttc aaa ttg gcc aaa tcc tgg ccc acc ctg aac atg cta 2592 Leu Arg Val Phe Lys Leu Ala Lys Ser Trp Pro Thr Leu Asn Met Leu 850 855 860 atc aag att att gga aat tca gtg ggt gcc ctg ggc aac ctg aca ctg 2640 Ile Lys Ile Ile Gly Asn Ser Val Gly Ala Leu Gly Asn Leu Thr Leu 865 870 875 880 gtg cta gcc att att gtc ttc atc ttt gcc gtg gtg ggg atg caa ctc 2688 Val Leu Ala Ile Ile Val Phe Ile Phe Ala Val Val Gly Met Gln Leu 885 890 895 ttt gga aaa agc tac aaa gag tgt gtc tgc aag atc aac cag gac tgt 2736 Phe Gly Lys Ser Tyr Lys Glu Cys Val Cys Lys Ile Asn Gln Asp Cys 900 905 910 gaa ctc cct cgc tgg cat atg cat gac ttt ttc cat tcc ttc ctc att 2784 Glu Leu Pro Arg Trp His Met His Asp Phe Phe His Ser Phe Leu Ile 915 920 925 gtc ttt cga gtg ttg tgc ggg gag tgg att gag acc atg tgg gac tgc 2832 Val Phe Arg Val Leu Cys Gly Glu Trp Ile Glu Thr Met Trp Asp Cys 930 935 940 atg gaa gtg gca ggc cag gcc atg tgc ctc att gtc ttt atg atg gtc 2880 Met Glu Val Ala Gly Gln Ala Met Cys Leu Ile Val Phe Met Met Val 945 950 955 960 atg gtg att ggc aac ttg gtg gtg ctg aac ctg ttt ctg gcc ttg ctc 2928 Met Val Ile Gly Asn Leu Val Val Leu Asn Leu Phe Leu Ala Leu Leu 965 970 975 ctg agc tcc ttc agt gca gac aac ctg gct gcc aca gat gac gat ggg 2976 Leu Ser Ser Phe Ser Ala Asp Asn Leu Ala Ala Thr Asp Asp Asp Gly 980 985 990 gaa atg aac aac ctc cag atc tca gtg atc cgt atc aag aag ggt gtg 3024 Glu Met Asn Asn Leu Gln Ile Ser Val Ile Arg Ile Lys Lys Gly Val 995 1000 1005 gcc tgg acc aaa cta aag gtg cac gcc ttc atg cag gcc cac ttt aag 3072 Ala Trp Thr Lys Leu Lys Val His Ala Phe Met Gln Ala His Phe Lys 1010 1015 1020 cag cgt gag gct gat gag gtg aag cct ctg gat gag ttg tat gaa aag 3120 Gln Arg Glu Ala Asp Glu Val Lys Pro Leu Asp Glu Leu Tyr Glu Lys 1025 1030 1035 1040 aag gcc aac tgt atc gcc aat cac acc ggt gca gac atc cac cgg aat 3168 Lys Ala Asn Cys Ile Ala Asn His Thr Gly Ala Asp Ile His Arg Asn 1045 1050 1055 ggt gac ttc cag aag aat ggc aat ggc aca acc agc ggc att ggc agc 3216 Gly Asp Phe Gln Lys Asn Gly Asn Gly Thr Thr Ser Gly Ile Gly Ser 1060 1065 1070 agc gtg gag aag tac atc att gat gag gac cac atg tcc ttc atc aac 3264 Ser Val Glu Lys Tyr Ile Ile Asp Glu Asp His Met Ser Phe Ile Asn 1075 1080 1085 aac ccc aac ttg act gta cgg gta ccc att gct gtg ggc gag tct gac 3312 Asn Pro Asn Leu Thr Val Arg Val Pro Ile Ala Val Gly Glu Ser Asp 1090 1095 1100 ttt gag aac ctc aac aca gag gat gtt agc agc gag tcg gat cct gaa 3360 Phe Glu Asn Leu Asn Thr Glu Asp Val Ser Ser Glu Ser Asp Pro Glu 1105 1110 1115 1120 ggc agc aaa gat aaa cta gat gac acc agc tcc tct gaa gga agc acc 3408 Gly Ser Lys Asp Lys Leu Asp Asp Thr Ser Ser Ser Glu Gly Ser Thr 1125 1130 1135 att gat atc aaa cca gaa gta gaa gag gtc cct gtg gaa cag cct gag 3456 Ile Asp Ile Lys Pro Glu Val Glu Glu Val Pro Val Glu Gln Pro Glu 1140 1145 1150 gaa tac ttg gat cca gat gcc tgc ttc aca gaa ggt tgt gtc cag cgg 3504 Glu Tyr Leu Asp Pro Asp Ala Cys Phe Thr Glu Gly Cys Val Gln Arg 1155 1160 1165 ttc aag tgc tgc cag gtc aac atc gag gaa ggg cta ggc aag tct tgg 3552 Phe Lys Cys Cys Gln Val Asn Ile Glu Glu Gly Leu Gly Lys Ser Trp 1170 1175 1180 tgg atc ctg cgg aaa acc tgc ttc ctc atc gtg gag cac aac tgg ttt 3600 Trp Ile Leu Arg Lys Thr Cys Phe Leu Ile Val Glu His Asn Trp Phe 1185 1190 1195 1200 gag acc ttc atc atc ttc atg att ctg ctg agc agt ggc gcc ctg gcc 3648 Glu Thr Phe Ile Ile Phe Met Ile Leu Leu Ser Ser Gly Ala Leu Ala 1205 1210 1215 ttc gag gac atc tac att gag cag aga aag acc atc cgc acc atc ctg 3696 Phe Glu Asp Ile Tyr Ile Glu Gln Arg Lys Thr Ile Arg Thr Ile Leu 1220 1225 1230 gaa tat gct gac aaa gtc ttc acc tat atc ttc atc ctg gag atg ttg 3744 Glu Tyr Ala Asp Lys Val Phe Thr Tyr Ile Phe Ile Leu Glu Met Leu 1235 1240 1245 ctc aag tgg aca gcc tat ggc ttc gtc aag ttc ttc acc aat gcc tgg 3792 Leu Lys Trp Thr Ala Tyr Gly Phe Val Lys Phe Phe Thr Asn Ala Trp 1250 1255 1260 tgt tgg ctg gac ttc ctc att gtg gct gtc tct tta gtc agc ctt ata 3840 Cys Trp Leu Asp Phe Leu Ile Val Ala Val Ser Leu Val Ser Leu Ile 1265 1270 1275 1280 gct aat gcc ctg ggc tac tcg gaa cta ggt gcc ata aag tcc ctt agg 3888 Ala Asn Ala Leu Gly Tyr Ser Glu Leu Gly Ala Ile Lys Ser Leu Arg 1285 1290 1295 acc cta aga gct ttg aga ccc tta aga gcc tta tca cga ttt gaa ggg 3936 Thr Leu Arg Ala Leu Arg Pro Leu Arg Ala Leu Ser Arg Phe Glu Gly 1300 1305 1310 atg agg gtg gtg gtg aat gcc ttg gtg ggc gcc atc ccc tcc atc atg 3984 Met Arg Val Val Val Asn Ala Leu Val Gly Ala Ile Pro Ser Ile Met 1315 1320 1325 aat gtg ctg ctg gtg tgt ctc atc ttc tgg ctg att ttc agc atc atg 4032 Asn Val Leu Leu Val Cys Leu Ile Phe Trp Leu Ile Phe Ser Ile Met 1330 1335 1340 gga gtt aac ttg ttt gcg gga aag tac cac tac tgc ttt aat gag act 4080 Gly Val Asn Leu Phe Ala Gly Lys Tyr His Tyr Cys Phe Asn Glu Thr 1345 1350 1355 1360 tct gaa atc cga ttt gaa att gaa gat gtc aac aat aaa act gaa tgt 4128 Ser Glu Ile Arg Phe Glu Ile Glu Asp Val Asn Asn Lys Thr Glu Cys 1365 1370 1375 gaa aag ctt atg gag ggg aac aat aca gag atc aga tgg aag aac gtg 4176 Glu Lys Leu Met Glu Gly Asn Asn Thr Glu Ile Arg Trp Lys Asn Val 1380 1385 1390 aag atc aac ttt gac aat gtt ggg gca gga tac ctg gcc ctt ctt caa 4224 Lys Ile Asn Phe Asp Asn Val Gly Ala Gly Tyr Leu Ala Leu Leu Gln 1395 1400 1405 gta gca acc ttc aaa ggc tgg atg gac atc atg tat gca gct gta gat 4272 Val Ala Thr Phe Lys Gly Trp Met Asp Ile Met Tyr Ala Ala Val Asp 1410 1415 1420 tcc cgg aag cct gat gag cag cct aag tat gag gac aat atc tac atg 4320 Ser Arg Lys Pro Asp Glu Gln Pro Lys Tyr Glu Asp Asn Ile Tyr Met 1425 1430 1435 1440 tac atc tat ttt gtc atc ttc atc atc ttc ggc tcc ttc ttc acc ctg 4368 Tyr Ile Tyr Phe Val Ile Phe Ile Ile Phe Gly Ser Phe Phe Thr Leu 1445 1450 1455 aac ctg ttc att ggt gtc atc att gat aac ttc aat caa caa aag aaa 4416 Asn Leu Phe Ile Gly Val Ile Ile Asp Asn Phe Asn Gln Gln Lys Lys 1460 1465 1470 aag ttc gga ggt cag gac atc ttc atg acc gaa gaa cag aag aag tac 4464 Lys Phe Gly Gly Gln Asp Ile Phe Met Thr Glu Glu Gln Lys Lys Tyr 1475 1480 1485 tac aat gcc atg aaa aag ctg ggc tca aag aag cca cag aaa cct att 4512 Tyr Asn Ala Met Lys Lys Leu Gly Ser Lys Lys Pro Gln Lys Pro Ile 1490 1495 1500 ccc cgc ccc ttg aac aaa atc caa gga atc gtc ttt gat ttt gtc act 4560 Pro Arg Pro Leu Asn Lys Ile Gln Gly Ile Val Phe Asp Phe Val Thr 1505 1510 1515 1520 cag caa gcc ttt gac att gtt atc atg atg ctc atc tgc ctt aac atg 4608 Gln Gln Ala Phe Asp Ile Val Ile Met Met Leu Ile Cys Leu Asn Met 1525 1530 1535 gtg aca atg atg gtg gag aca gac act caa agc aag cag atg gag aac 4656 Val Thr Met Met Val Glu Thr Asp Thr Gln Ser Lys Gln Met Glu Asn 1540 1545 1550 atc ctc tac tgg att aac ctg gtg ttt gtt atc ttc ttc acc tgt gag 4704 Ile Leu Tyr Trp Ile Asn Leu Val Phe Val Ile Phe Phe Thr Cys Glu 1555 1560 1565 tgt gtg ctc aaa atg ttt gcg ttg agg cac tac tac ttc acc att ggc 4752 Cys Val Leu Lys Met Phe Ala Leu Arg His Tyr Tyr Phe Thr Ile Gly 1570 1575 1580 tgg aac atc ttc gac ttc gtg gta gtc atc ctc tcc att gtg gga atg 4800 Trp Asn Ile Phe Asp Phe Val Val Val Ile Leu Ser Ile Val Gly Met 1585 1590 1595 1600 ttc ctg gca gat ata att gag aaa tac ttt gtt tcc cca acc cta ttc 4848 Phe Leu Ala Asp Ile Ile Glu Lys Tyr Phe Val Ser Pro Thr Leu Phe 1605 1610 1615 cga gtc atc cga ttg gcc cgt att ggg cgc atc ttg cgt ctg atc aaa 4896 Arg Val Ile Arg Leu Ala Arg Ile Gly Arg Ile Leu Arg Leu Ile Lys 1620 1625 1630 ggc gcc aaa ggg att cgt acc ctg ctc ttt gcc tta atg atg tcc ttg 4944 Gly Ala Lys Gly Ile Arg Thr Leu Leu Phe Ala Leu Met Met Ser Leu 1635 1640 1645 cct gcc ctg ttc aac atc ggc ctt ctg ctc ttc ctg gtc atg ttc atc 4992 Pro Ala Leu Phe Asn Ile Gly Leu Leu Leu Phe Leu Val Met Phe Ile 1650 1655 1660 ttc tcc att ttt ggg atg tcc aat ttt gca tat gtg aag cac gag gct 5040 Phe Ser Ile Phe Gly Met Ser Asn Phe Ala Tyr Val Lys His Glu Ala 1665 1670 1675 1680 ggt atc gat gac atg ttc aac ttt gag aca ttt ggc aac agc atg atc 5088 Gly Ile Asp Asp Met Phe Asn Phe Glu Thr Phe Gly Asn Ser Met Ile 1685 1690 1695 tgc ctg ttt caa atc gca acc tca gct ggt tgg gat ggc ctg ctg ctg 5136 Cys Leu Phe Gln Ile Ala Thr Ser Ala Gly Trp Asp Gly Leu Leu Leu 1700 1705 1710 ccc atc cta aac cgc ccc cct gac tgc agc cta gat aag gaa cac cca 5184 Pro Ile Leu Asn Arg Pro Pro Asp Cys Ser Leu Asp Lys Glu His Pro 1715 1720 1725 ggg agt ggc ttt aag gga gat tgt ggg aac ccc tca gtg ggc atc ttc 5232 Gly Ser Gly Phe Lys Gly Asp Cys Gly Asn Pro Ser Val Gly Ile Phe 1730 1735 1740 ttc ttt gta agc tac atc atc atc tct ttc cta att gtc gtg aac atg 5280 Phe Phe Val Ser Tyr Ile Ile Ile Ser Phe Leu Ile Val Val Asn Met 1745 1750 1755 1760 tac att gcc atc atc ctg gag aac ttc agt gta gcc aca gag gaa agt 5328 Tyr Ile Ala Ile Ile Leu Glu Asn Phe Ser Val Ala Thr Glu Glu Ser 1765 1770 1775 gca gac cct ctg agt gag gat gac ttt gag acc ttc tat gag atc tgg 5376 Ala Asp Pro Leu Ser Glu Asp Asp Phe Glu Thr Phe Tyr Glu Ile Trp 1780 1785 1790 gag aag ttc gac ccc gat gcc acc cag ttc att gag tac tgt aag ctg 5424 Glu Lys Phe Asp Pro Asp Ala Thr Gln Phe Ile Glu Tyr Cys Lys Leu 1795 1800 1805 gca gac ttt gca gat gcc ttg gag cat cct ctc cga gtg ccc aag ccc 5472 Ala Asp Phe Ala Asp Ala Leu Glu His Pro Leu Arg Val Pro Lys Pro 1810 1815 1820 aat acc att gag ctc atc gct atg gat ctg cca atg gtg agc ggg gat 5520 Asn Thr Ile Glu Leu Ile Ala Met Asp Leu Pro Met Val Ser Gly Asp 1825 1830 1835 1840 cgc atc cac tgc ttg gac atc ctt ttt gcc ttc acc aag cgg gtc ctg 5568 Arg Ile His Cys Leu Asp Ile Leu Phe Ala Phe Thr Lys Arg Val Leu 1845 1850 1855 gga gat agc ggg gag ttg gac atc ctg cgg cag cag atg gaa gag cgg 5616 Gly Asp Ser Gly Glu Leu Asp Ile Leu Arg Gln Gln Met Glu Glu Arg 1860 1865 1870 ttc gtg gca tcc aat cct tcc aaa gtg tct tac gag cca atc aca acc 5664 Phe Val Ala Ser Asn Pro Ser Lys Val Ser Tyr Glu Pro Ile Thr Thr 1875 1880 1885 aca ctg cgt cgc aag cag gag gag gta tct gca gtg gtc ctg cag cgt 5712 Thr Leu Arg Arg Lys Gln Glu Glu Val Ser Ala Val Val Leu Gln Arg 1890 1895 1900 gcc tac cgg gga cat ttg gca agg cgg ggc ttc atc tgc aaa aag aca 5760 Ala Tyr Arg Gly His Leu Ala Arg Arg Gly Phe Ile Cys Lys Lys Thr 1905 1910 1915 1920 act tct aat aag ctg gag aat gga ggc aca cac cgg gag aaa aaa gag 5808 Thr Ser Asn Lys Leu Glu Asn Gly Gly Thr His Arg Glu Lys Lys Glu 1925 1930 1935 agc acc cca tct aca gcc tcc ctc ccg tcc tat gac agt gta act aaa 5856 Ser Thr Pro Ser Thr Ala Ser Leu Pro Ser Tyr Asp Ser Val Thr Lys 1940 1945 1950 cct gaa aag gag aaa cag cag cgg gca gag gaa gga aga agg gaa aga 5904 Pro Glu Lys Glu Lys Gln Gln Arg Ala Glu Glu Gly Arg Arg Glu Arg 1955 1960 1965 gcc aaa aga caa aaa gag gtc aga gaa tcc aag tgt tagaggagaa 5950 Ala Lys Arg Gln Lys Glu Val Arg Glu Ser Lys Cys 1970 1975 1980 caaaaattca gtattataca gatctaaaac tcgcaagtga aagattgttt acaaacttcc 6010 tgaatattat caatgcagaa cagctgtgga gactctaacc tgaagatcta taccaaacgt 6070 cgtctgctta ccacgtaaca cagctgcatc ttgagcagtg acctccaagg gcaaagtacc 6130 ccgctcccta gacttacaga ttttctaatg cttgggcagg tggttactgc atgttccaca 6190 tcagtcaatg caacttagga caaaactaac cagatacaga aacagaagag aggctgccgg 6250 gaccagcata tttccgttgc agccaaatgg attttatttt ttcattttat tgattctcag 6310 aagcagaaag catcacttta aaagttcgtt tgttcatgca aactatattt gcattcttac 6370 attagttaag ctaagcagca aaaagaaaac acacacacac actcacattt agcccatgtc 6430 atttaattgt cagtttcttt gacataaagc gcatcttctc cacatgggct tcacgtggtt 6490 tggagatggg tgggggaaaa caatcaggtt tcttcaggct gaggaggact tgctcaggcc 6550 gattccaaac attgtgctcg ttcaatgcgt agaaatgatt tgcatgatgg catgccgtga 6610 tcagaagtca tgcatgagat ccatacacca caggacacta ctaatctagt cccttgcact 6670 gggtcagcct ttggacagga cccagccctg caccgttcac tgtatttgga gaaaatggta 6730 agagttccat accggctaca attctttgag ttcttaaaag tccttcatac accttctggg 6790 tagggaaaca accaactaat tgactaacac caccaacaac aaaaaacaaa cccaatccaa 6850 caagcagatg gatccgttgc gtgtatatgt ttaacagaca tctccaacat acagccattg 6910 ttgcacattt tgcaagatga actatttaat gctgctctgt gtccagtaca tgggggagac 6970 tttgatccca aatggcttgt actatttatg tcactgtaaa accaaatcct agggctaaaa 7030 aaaaaaaaaa aaaaaaaaaa aaa 7053[Sequence List] SEQUENCE LISTING <110> Japan Science and Technology Agency <120> Sodium Channel SCN8A <160> 1 <170> PatentIn Ver. 2.0 <210> 1 <211> 7053 <212> DNA <213> Homo sapiens <220 > <221> CDS <222> (1) .. (5940) <400> 1 atg gcg gcg cgg gtg ctt gca cca cca ggc cct gat agt ttc aag cct 48 Met Ala Ala Arg Val Leu Ala Pro Pro Gly Pro Asp Ser Phe Lys Pro 1 5 10 15 ttc acc cct gag tca ctg gca aac att gag agg cgc att gct gag agc 96 Phe Thr Pro Glu Ser Leu Ala Asn Ile Glu Arg Arg Ile Ala Glu Ser 20 25 30 aag ctc aag aaa cca cca aag gcc gat ggc agt cat cgg gag gac gat 144 Lys Leu Lys Lys Pro Pro Lys Ala Asp Gly Ser His Arg Glu Asp Asp 35 40 45 gag gac agc aag ccc aag cca aac agc gac ctg gaa gca ggg aag agt 192 Glu Asp Ser Lys Pro Lys Pro Asn Ser Asp Leu Glu Ala Gly Lys Ser 50 55 60 ttg cct ttc atc tac ggg gac atc ccc caa ggc ctg gtt gca gtt ccc 240 Leu Pro Phe Ile Tyr Gly Asp Ile Pro Gln Gly Leu Val Ala Val Pro 65 70 75 80 ctg gag gac ttt gac cca tac tat ttg acg cag aaa acc ttt gta gta 288 Leu Glu Asp Phe Asp Pro Tyr Tyr Leu Thr Gln Lys Thr Phe Val Val 85 90 95 tta aac aga ggg aaa act ctc ttc aga ttt agt gcc acg cct gcc ttg 336 Leu Asn Arg Gly Lys Thr Leu Phe Arg Phe Ser Ala Thr Pro Ala Leu 100 105 110 tac att tta agt cct ttt aac ctg ata aga aga ata gct att aaa att 384 Tyr Ile Leu Ser Pro Phe Asn Leu Ile Arg Arg Ile Ala Ile Lys Ile 115 120 125 ttg ata cat tca gta ttt agc atg atc att atg tgc act att ttg acc 432 Leu Ile His Ser Val Phe Ser Met Ile Ile Met Cys Thr Ile Leu Thr 130 135 140 aac tgt gta ttc atg act ttt agt aac cct cct gac tgg tcg aag aat 480 Asn Cys Val Phe Met Thr Phe Ser Asn Pro Pro Asp Trp Ser Lys Asn 145 150 155 160 gtg gag tac acg ttc aca ggg att tat aca ttt gaa tca cta gtg aaa 528 Val Glu Tyr Thr Phe Thr Gly Ile Tyr Thr Phe Glu Ser Leu Val Lys 165 170 175 atc att gca aga ggt ttc tgc ata gat ggc ttt acc ttt tta cgg gat 576 Ile Ile Ala Arg Gly Phe Cys Ile Asp Gly Phe Thr Phe Leu Arg Asp 180 185 190 cca tgg aac tgg tta gat ttc agt gt atg atg gcg tat ata aca 624 Pro Trp Asn Trp Leu Asp Phe Ser Val Ile Met Met Ala Tyr Ile Thr 195 200 205 gag ttt gta aac cta ggc aat gtt tca gct cta cgc act ttc agg gta 672 Glu Phe Val Asn Leu Gly Asn Val Ser Ala Leu Arg Thr Phe Arg Val 210 215 220 ctg agg gct ttg aaa act att tcg gta atc cca ggc ctg aag aca att 720 Leu Arg Ala Leu Lys Thr Ile Ser Val Ile Pro Gly Leu Lys Thr Ile 225 230 235 240 gtg ggt gcc ctg att cag tct gtg aag aaa ctg tca gat gtg atg atc 768 Val Gly Ala Leu Ile Gln Ser Val Lys Lys Leu Ser Asp Val Met Ile 245 250 255 atg aca gtg ttc tgc ctg agt gtt ttt gcc ttg atg gct 816 Met Thr Val Phe Cys Leu Ser Val Phe Ala Leu Ile Gly Leu Gln Leu 260 265 270 ttt cat ggg gaa cct tcg aac aag tgt gtt gtg tgg ccc ata aac ttc 864 Phe His Gly Glu Pro Ser Asn Lys Cys Val Val Trp Pro Ile Asn Phe 275 280 285 aac gag agc tat ctt gaa aat ggc acc aaa ggc ttt gat tgg gaa gag 912 Asn Glu Ser Tyr Leu Glu Asn Gly Thr Lys Gly Phe Asp Trp Glu Glu 290 295 300 tat atc aac aat aaa aca c tac aca gtt cct ggc atg ctg gaa 960 Tyr Ile Asn Asn Lys Thr Asn Phe Tyr Thr Val Pro Gly Met Leu Glu 305 310 315 320 cct tta ctc tgt ggg aac agt tct gat gct ggg caa tgc cca gag gga 1008 Pro Leu Leu Cys Gly Asn Ser Ser Asp Ala Gly Gln Cys Pro Glu Gly 325 330 335 tac cag tgt atg aaa gca gga agg aac ccc aac tat ggt tac aca agt 1056 Tyr Gln Cys Met Lys Ala Gly Arg Asn Pro Asn Tyr Gly Tyr Thr Ser 340 345 350 ttt gac act ttt agc tgg gcc ttc ttg gca tta ttt cgc ctt atg acc 1104 Phe Asp Thr Phe Ser Trp Ala Phe Leu Ala Leu Phe Arg Leu Met Thr 355 360 365 cag gac tat tgg gaa aac ttg tat caa ttg act cga gca gcc ggg 1152 Gln Asp Tyr Trp Glu Asn Leu Tyr Gln Leu Thr Leu Arg Ala Ala Gly 370 375 380 aaa aca tac atg atc ttc ttc gtc ttg gtc atc ttt gtg ggt tct ttc 1200 Lys Thr Tyr Val Leu Phe Val Ile Phe Val Gly Ser Phe 385 390 395 400 tat ctg gtg aac ttg atc ttg gct gtg gtg gcc atg gct tat gaa gaa 1248 Tyr Leu Val Asn Leu Ile Leu Ala Val Val Ala Met Ala Tyr Glu Glu 405 410 415 cag at cag gca aca ctg gag gag gca gaa caa aaa gag gct gaa ttt 1296 Gln Asn Gln Ala Thr Leu Glu Glu Ala Glu Gln Lys Glu Ala Glu Phe 420 425 430 aaa gca atg ttg gag caa ctt aag aag caa gag gag gag 1344 Lys Ala Met Leu Glu Gln Leu Lys Lys Gln Gln Glu Glu Ala Gln Ala 435 440 445 gct gcg atg gcc aat tca gca gga act gtc tca gaa gat gcc ata gag 1392 Ala Ala Met Ala Asn Ser Ala Gly Thr Val Ser Glu Ala Ile Glu 450 455 460 gaa gaa ggt gaa gaa gga ggg ggc tcc cct cgg agc ttt tct gaa atc 1440 Glu Glu Glu Gly Glu Glu Gly Gly Gly Ser Pro Arg Ser Phe Ser Glu Ile 465 470 475 475 480 tct aaa atcagc gca aag gaa aga agt aac agg aga aag 1488 Ser Lys Ile Ser Ser Lys Ser Ala Lys Glu Arg Ser Asn Arg Arg Lys 485 490 495 aag agg aag caa aag gaa ctc ttt gaa gga gag gag aaa ggg gat ccc 1536 Lys Arg Lys Lys Glu Leu Phe Glu Gly Glu Glu Lys Gly Asp Pro 500 505 510 gag aag gtg ttt aag tca gag tca gaa gat ggc atg aga agg aag gcc 1584 Glu Lys Val Phe Lys Ser Glu Ser Glu Asp Gly Met Arg Arg Lys Ala 5 15 520 525 ttt cgg ctg cca gac aac aga ata ggg agg aaa ttt tcc atc atg aat 1632 Phe Arg Leu Pro Asp Asn Arg Ile Gly Arg Lys Phe Ser Ile Met Asn 530 535 540 cag tca atg ttc agc atc cca ccctc ctc tcc cgc cac aac 1680 Gln Ser Met Phe Ser Ile Pro Gly Ser Pro Phe Leu Ser Arg His Asn 545 550 555 560 agc aag agc agc atc ttc agt ttc agg gga cct ggg cgg ttc cga gac 1728 Ser Lys Ser Ser Ile Phe Ser Phe Arg Gly Pro Gly Arg Phe Arg Asp 565 570 575 ccg ggc tcc gag aat gag ttc gcg gat gac gag cac agc acg gtg gag 1776 Pro Gly Ser Glu Asn Glu Phe Ala Asp Asp Glu His Ser Thr Val Glu 580 585 g 590 gag gag ggc cgc cgg gac tcc ctc ttc atc ccc atc cgg gcc cgc 1824 Glu Ser Glu Gly Arg Arg Asp Ser Leu Phe Ile Pro Ile Arg Ala Arg 595 600 600 605 gag cgc cgg agc agc tac agc ggc tac agcg cg agcg gc tac agcg cg agcgc gac tg 1872 Glu Arg Arg Ser Ser Tyr Ser Gly Tyr Ser Gly Tyr Ser Gln Gly Ser 610 615 620 cgc tcc tcg cgc atc ttc ccc agc ctg cgg cgc agc gtg aag cgc aac 1920 Arg Ser Ser Arg Ile Phe Pro Ser Leu Arg Arg Se r Val Lys Arg Asn 625 630 635 640 agc acg gtg gac tgc aac ggc gtg gtg tcc ctc atc ggc ggc ccc ggc 1968 Ser Thr Val Asp Cys Asn Gly Val Val Ser Leu Ile Gly Gly Pro Gly 645 650 655 ttc cgg atc cgt ctc ctg cca gag gct aca act gag gtg gaa 2016 Ser His Ile Gly Gly Arg Leu Leu Pro Glu Ala Thr Thr Glu Val Glu 660 665 670 att aag aag aaa ggc cct gga tct ctt tta gtt tcc atg gac caa tta 2064 Ile Lys Lys Lys Gly Pro Gly Ser Leu Leu Val Ser Met Asp Gln Leu 675 680 685 gcc tcc tac ggg cgg aag gac aga atc aac agt ata atg agt gtt gtt 2112 Ala Ser Tyr Gly Arg Lys Asp Arg Ile Asn Ser Ile Met Ser Val Val 690 695 700 aca aat aca cta gta gaa gaa ctg gaa gag tct cag aga aag tgc ccg 2160 Thr Asn Thr Leu Val Glu Glu Leu Glu Glu Ser Gln Arg Lys Cys Pro 705 710 715 720 cca tgc tgg tat aaa ttt gcc aac act ctc atc tgg gag tgc cac 2208 Pro Cys Trp Tyr Lys Phe Ala Asn Thr Phe Leu Ile Trp Glu Cys His 725 730 735 ccc tac tgg ata aaa ctg aaa gag att gtg aac ttg ata gtt atg gac 2256 Pro Tyr Lep Lys u Lys Glu Ile Val Asn Leu Ile Val Met Asp 740 745 750 cct ttt gtg gat tta gcc atc acc atc tgc atc gtc ctg aat aca ctg 2304 Pro Phe Val Asp Leu Ala Ile Thr Ile Cys Ile Val Leu Asn Thr Leu 755 760 765 ttt atg gca atg gag cac cat cct atg aca cca caa ttt gaa cat gtc 2352 Phe Met Ala Met Glu His His Pro Met Thr Pro Gln Phe Glu His Val 770 775 780 ttg gct gta gga aat ctg gtt ttc act gga att ttc aca gg gaa atg 2400 Leu Ala Val Gly Asn Leu Val Phe Thr Gly Ile Phe Thr Ala Glu Met 785 790 795 800 ttc ctg aag ctc ata gcc atg gat ccc tac tat tat ttc caa gaa ggt 2448 Phe Leu Lys Leu Ile Ala Met Asp Tyr Tyr Phe Gln Glu Gly 805 810 815 tgg aac att ttt gac gga ttt att gtc tcc ctc agt tta atg gaa ctg 2496 Trp Asn Ile Phe Asp Gly Phe Ile Val Ser Leu Ser Leu Met Glu Leu 820 825 830 agt cg gca gca gag ggg ctt tca gtg ctg cga tct ttc cga ttg 2544 Ser Leu Ala Asp Val Glu Gly Leu Ser Val Leu Arg Ser Phe Arg Leu 835 840 845 ctc cga gtc ttc aaa ttg gcc aaa tcc tgg ccc acc ctg aac atgta 2 Arg Val Phe Lys Leu Ala Lys Ser Trp Pro Thr Leu Asn Met Leu 850 855 860 atc aag att att gga aat tca gtg ggt gcc ctg ggc aac ctg aca ctg 2640 Ile Lys Ile Ile Gly Asn Ser Val Gly Ala Leu Gly Asn Leu Thr Leu 865 870 875 880 gtg cta gcc att att gtc ttc atc ttt gcc gtg gtg ggg atg caa ctc 2688 Val Leu Ala Ile Ile Val Phe Ile Phe Ala Val Val Gly Met Gln Leu 885 890 895 895 ttt gga aaa agc tacgt g g tgc aag atc aac cag gac tgt 2736 Phe Gly Lys Ser Tyr Lys Glu Cys Val Cys Lys Ile Asn Gln Asp Cys 900 905 910 gaa ctc cct cgc tgg cat atg cat gac ttt ttc cat tcc ttc ctc att 2784 Glu Leu Pro Arg Trp Met His Asp Phe Phe His Ser Phe Leu Ile 915 920 925 gtc ttt cga gtg ttg tgc ggg gag tgg att gag acc atg tgg gac tgc 2832 Val Phe Arg Val Leu Cys Gly Glu Trp Ile Glu Thr Met Trp Asp Cys 930 935 940g gaa gtg gca ggc cag gcc atg tgc ctc att gtc ttt atg atg gtc 2880 Met Glu Val Ala Gly Gln Ala Met Cys Leu Ile Val Phe Met Met Val 945 950 955 960 atg gtg att ggc aac ttg ctg gtg gtg gtg gtg gtg gtg gtg gtg gtg gtg gtg gtg gtg ctg tg gcc ttg ctc 2928 Met Val Ile Gly Asn Leu Val Val Leu Asn Leu Phe Leu Ala Leu Leu 965 970 975 ctg agc tcc ttc agt gca gac aac ctg gct gcc aca gat gac gat ggg 2976 Leu Ser Ser Phe Le Ala Asp As Ala Ala Thr Asp Asp Asp Gly 980 985 990 gaa atg aac aac ctc cag atc tca gtg atc cgt atc aag aag ggt gtg 3024 Glu Met Asn Asn Leu Gln Ile Ser Val Ile Arg Ile Lys Lys Gly Val 995 1000 1005 gcc tgg acc aaa cta aag gtg cac gcc ttc atg cag gcc cac ttt aag 3072 Ala Trp Thr Lys Leu Lys Val His Ala Phe Met Gln Ala His Phe Lys 1010 1015 1020 cag cgt gag gct gat gag gtg aag cct ctg gat gag ttg 3 gat ag Arg Glu Ala Asp Glu Val Lys Pro Leu Asp Glu Leu Tyr Glu Lys 1025 1030 1035 1040 aag gcc aac tgt atc gcc aat cac acc ggt gca gac atc cac cgg aat 3168 Lys Ala Asn Cys Ile Ala Asn His Thr Gly Ala Asp Ile His Arg Asn 1045 1050 1055 ggt gac ttc cag aag aat ggc aat ggc aca acc agc ggc att ggc agc 3216 Gly Asp Phe Gln Lys Asn Gly Asn Gly Thr Thr Ser Gly Ile Gly Ser 1060 1065 1070 agc gtg gag aa g tac atc att gat gag gac cac atg tcc ttc atc aac 3264 Ser Val Glu Lys Tyr Ile Ile Asp Glu Asp His Met Ser Phe Ile Asn 1075 1080 1085 aac ccc aac ttg act gta cgg gta ccc att gct gtg ggc gag tct gac 3312 Asn Pro Asn Leu Thr Val Arg Val Pro Ile Ala Val Gly Glu Ser Asp 1090 1095 1100 ttt gag aac ctc aac aca gag gat gtt agc agc gag tcg gat cct gaa 3360 Phe Glu Asn Leu Asn Thr Glu Asp Val Ser Ser Glu Ser Asp Pro Glu 1105 1110 1115 1120 ggc agc aaa gat aaa cta gat gac acc agc tcc tct gaa gga agc acc 3408 Gly Ser Lys Asp Lys Leu Asp Asp Thr Ser Ser Ser Glu Gly Ser Thr 1125 1130 1135 att gat atc aaa cca gaa gtagaa gag gtc cct gtg gaa cag cct gag 3456 Ile Asp Ile Lys Pro Glu Val Glu Glu Val Pro Val Glu Gln Pro Glu 1140 1145 1150 gaa tac ttg gat cca gat gcc tgc ttc aca gaa ggt tgt gtc cag cgg 3504 Glu Tyr Asp Ala Cys Phe Thr Glu Gly Cys Val Gln Arg 1155 1160 1165 ttc aag tgc tgc cag gtc aac atc gag gaa ggg cta ggc aag tct tgg 3552 Phe Lys Cys Cys Gln Val Asn Ile Glu Glu Glu Gly Leu Gly Lys Ser Trp 1170 1175 1180 tgg atc ctg cgg aaa acc tgc ttc ctc atc gtg gag cac aac tgg ttt 3600 Trp Ile Leu Arg Lys Thr Cys Phe Leu Ile Val Glu His Asn Trp Phe 1185 1190 1195 1200 gag acc ttc atg atc atc ttc att ctg ctg agc agt ggc gcc ctg gcc 3648 Glu Thr Phe Ile Ile Phe Met Ile Leu Leu Ser Ser Gly Ala Leu Ala 1205 1210 1215 ttc gag gac atc tac att gag cag aga aag acc atc cgc acc atc ctg 3696 Phe Ghe Tyr Ile Glu Gln Arg Lys Thr Ile Arg Thr Ile Leu 1220 1225 1230 gaa tat gct gac aaa gtc ttc acc tat atc ttc atc ctg gag atg ttg 3744 Glu Tyr Ala Asp Lys Val Phe Thr Tyr Ile Phe Ile Leu Glu Met Leu 1235 1245 ctc aag tgg aca gcc tat ggc ttc gtc aag ttc ttc acc aat gcc tgg 3792 Leu Lys Trp Thr Ala Tyr Gly Phe Val Lys Phe Phe Thr Asn Ala Trp 1250 1255 1260 tgt tgg ctg gac ttc ctc gtc gtg gt agc ctt ata 3840 Cys Trp Leu Asp Phe Leu Ile Val Ala Val Ser Leu Val Ser Leu Ile 1265 1270 1275 1280 gct aat gcc ctg ggc tac tcg gaa cta ggt gcc ata aag tcc ctt agg 3888 Ala As n Ala Leu Gly Tyr Ser Glu Leu Gly Ala Ile Lys Ser Leu Arg 1285 1290 1295 acc cta aga gct ttg aga ccc tta aga gcc tta tca cga ttt gaa ggg 3936 Thr Leu Arg Ala Leu Arg Pro Leu Arg Ala Leu Ser Arg Phe Glu Gly 1300 1305 1310 atg agg gtg gtg gtg aat gcc ttg gtg ggc gcc atc ccc tcc atc atg 3984 Met Arg Val Val Val Asn Ala Leu Val Gly Ala Ile Pro Ser Ile Met 1315 1320 1325 aat gtg ctg ctg gtg tgt gtg tgt ctg att ttc agc atc atg 4032 Asn Val Leu Leu Val Cys Leu Ile Phe Trp Leu Ile Phe Ser Ile Met 1330 1335 1340 gga gtt aac ttg ttt gcg gga aag tac cac tac tgc ttt aat gag act 4080 Gly Val Asn Leu Phe Lys Tyr His Tyr Cys Phe Asn Glu Thr 1345 1350 1355 1360 tct gaa atc cga ttt gaa att gaa gat gtc aac aat aaa act gaa tgt 4128 Ser Glu Ile Arg Phe Glu Ile Glu Asp Val Asn Asn Lys Thr Glu Cys 1365 1370 1375 aag ctt atg gag ggg aac aat aca gag atc aga tgg aag aac gtg 4176 Glu Lys Leu Met Glu Gly Asn Asn Thr Glu Ile Arg Trp Lys Asn Val 1380 1385 1390 aag atc aac ttt gac aat gtt ggg gc a gga tac ctg gcc ctt ctt caa 4224 Lys Ile Asn Phe Asp Asn Val Gly Ala Gly Tyr Leu Ala Leu Leu Gln 1395 1400 1405 gta gca acc ttc aaa ggc tgg atg gac atc atg tat gca gct gta gat 4272 Val Ala Thr Phe Gly Trp Met Asp Ile Met Tyr Ala Ala Val Asp 1410 1415 1420 tcc cgg aag cct gat gag cag cct aag tat gag gac aat atc tac atg 4320 Ser Arg Lys Pro Asp Glu Gln Pro Lys Tyr Glu Asp Asn Ile Tyr Met 1425 1430 1435 1440 tac atc tat ttt gtc atc ttc atc atc ttc ggc tcc ttc ttc acc ctg 4368 Tyr Ile Tyr Phe Val Ile Phe Ile Ile Phe Gly Ser Phe Phe Thr Leu 1445 1450 1455 aac ctg ttc att ggt gtc atc atc att atg caa aag aaa 4416 Asn Leu Phe Ile Gly Val Ile Ile Asp Asn Phe Asn Gln Gln Lys Lys 1460 1465 1470 aag ttc gga ggt cag gac atc ttc atg acc gaa gaa cag aag aag tac 4464 Lys Phe Gly Gly Gln Asp Ile Phe Glu Glu Gln Lys Lys Tyr 1475 1480 1485 tac aat gcc atg aaa aag ctg ggc tca aag aag cca cag aaa cct att 4512 Tyr Asn Ala Met Lys Lys Leu Gly Ser Lys Lys Pro Gln Lys Pro Ile 1490 149 5 1500 ccc cgc ccc ttg aac aaa atc caa gga atc gtc ttt gat ttt gtc act 4560 Pro Arg Pro Leu Asn Lys Ile Gln Gly Ile Val Phe Asp Phe Val Thr 1505 1510 1515 1520 cag caa gcc ttt gac att gtt atc atg atg ctc atc tgc ctt aac atg 4608 Gln Gln Ala Phe Asp Ile Val Ile Met Met Leu Ile Cys Leu Asn Met 1525 1530 1535 gtg aca atg atg gtg gag aca gac act caa agc aag cag atg gag aac 4656 Val Thr Met Met Val Glu Thr As Thr Gln Ser Lys Gln Met Glu Asn 1540 1545 1550 atc ctc tac tgg att aac ctg gtg ttt gtt atc ttc ttc acc tgt gag 4704 Ile Leu Tyr Trp Ile Asn Leu Val Phe Val Ile Phe Phe Thr Cys Glu 1555 1560 1565 tgt gt aaa atg ttt gcg ttg agg cac tac tac ttc acc att ggc 4752 Cys Val Leu Lys Met Phe Ala Leu Arg His Tyr Tyr Phe Thr Ile Gly 1570 1575 1580 tgg aac atc ttc gac ttc gtg gta gtc atc 4 gtc gcc atg Trp Asn Ile Phe Asp Phe Val Val Val Ile Leu Ser Ile Val Gly Met 1585 1590 1595 1600 ttc ctg gca gat ata att gag aaa tac ttt gtt tcc cca acc cta ttc 4848 Phe Leu Ala Asp Ile Ile G lu Lys Tyr Phe Val Ser Pro Thr Leu Phe 1605 1610 1615 cga gtc atc cga ttg gcc cgt att ggg cgc atc ttg cgt ctg atc aaa 4896 Arg Val Ile Arg Leu Ala Arg Ile Gly Arg Ile Leu Arg Leu Ile Lys 1620 1625 1630 gcc aaa ggg att cgt acc ctg ctc ttt gcc tta atg atg tcc ttg 4944 Gly Ala Lys Gly Ile Arg Thr Leu Leu Phe Ala Leu Met Met Ser Leu 1635 1640 1645 cct gcc ctg ttc aac atc ggc ctt ctg ctc gtc ttc ttc ctc ctc ctc gtc ttc ctc ttc ttc ttc atc 4992 Pro Ala Leu Phe Asn Ile Gly Leu Leu Leu Phe Leu Val Met Phe Ile 1650 1655 1660 ttc tcc att ttt ggg atg tcc aat ttt gca tat gtg aag cac gag gct 5040 Phe Ser Ile Phe Gly Met Ser Asn Phe Ala Tyr Val Lys His Glu Ala 1665 1670 1675 1680 ggt atc gat gac atg ttc aac ttt gag aca ttt ggc aac agc atg atc 5088 Gly Ile Asp Asp Met Phe Asn Phe Glu Thr Phe Gly Asn Ser Met Ile 1685 1690 1695 tgc ctg gtta ca acc tca gct ggt tgg gat ggc ctg ctg ctg 5136 Cys Leu Phe Gln Ile Ala Thr Ser Ala Gly Trp Asp Gly Leu Leu Leu 1700 1705 1710 ccc atc cta aac cgc ccc cct gac tgc agc cta gat aag g aa cac cca 5184 Pro Ile Leu Asn Arg Pro Pro Asp Cys Ser Leu Asp Lys Glu His Pro 1715 1720 1725 ggg agt ggc ttt aag gga gat tgt ggg aac ccc tca gtg ggc atc ttc 5232 Gly Ser Gly Phe Lys Gly Asp Cys Gly Asn Pro Ser Val Gly Ile Phe 1730 1735 1740 ttc ttt gta agc tac atc atc atc tct ttc cta att gtc gtg aac atg 5280 Phe Phe Val Ser Tyr Ile Ile Ile Ser Phe Leu Ile Val Val Asn Met 1745 1750 1755 1760 tac att gcc atc atc ctg gag aac ttc agt gta gcc aca gag gaa agt 5328 Tyr Ile Ala Ile Ile Leu Glu Asn Phe Ser Val Ala Thr Glu Glu Ser 1765 1770 1775 gca gac cct ctg agt gag gat gac ttt gag acc ttc tat gag atc tgg 5376 Asp Pro Leu Ser Glu Asp Asp Phe Glu Thr Phe Tyr Glu Ile Trp 1780 1785 1790 gag aag ttc gac ccc gat gcc acc cag ttc att gag tac tgt aag ctg 5424 Glu Lys Phe Asp Pro Asp Ala Thr Gln Phe Ile Glu Tyr Cys Lys Leu 1795 1800 1805 gca gac ttt gca gat gcc ttg gag cat cct ctc cga gtg ccc aag ccc 5472 Ala Asp Phe Ala Asp Ala Leu Glu His Pro Leu Arg Val Pro Lys Pro 1810 1815 1820 aat acc att gag ctc atc gct atg gat ctg cca atg gtg agc ggg gat 5520 Asn Thr Ile Glu Leu Ile Ala Met Asp Leu Pro Met Val Ser Gly Asp 1825 1830 1835 1840 cgc atc cac tgc ttg gac atc ctt ttt gcc ttc acc ag 5568 Arg Ile His Cys Leu Asp Ile Leu Phe Ala Phe Thr Lys Arg Val Leu 1845 1850 1855 gga gat agc ggg gag ttg gac atc ctg cgg cag cag atg gaa gag cgg 5616 Gly Asp Ser Gly Glu Leu Asp Ile Leu Arg Gln Mln Glu Glu Arg 1860 1865 1870 ttc gtg gca tcc aat cct tcc aaa gtg tct tac gag cca atc aca acc 5664 Phe Val Ala Ser Asn Pro Ser Lys Val Ser Tyr Glu Pro Ile Thr Thr 1875 1880 1885 aca ctg cgt cgc aag cag gag gag gta tct gca gtg gtc ctg cag cgt 5712 Thr Leu Arg Arg Lys Gln Glu Glu Val Ser Ala Val Val Leu Gln Arg 1890 1895 1900 gcc tac cgg gga cat ttg gca agg cgg ggc ttc atc tgc aaa aag aca 5760 Ala Tyr Arg Leu Ala Arg Arg Gly Phe Ile Cys Lys Lys Thr 1905 1910 1915 1920 act tct aat aag ctg gag aat gga ggc aca cac cgg gag aaa aaa gag 5808 Thr Ser Asn Lys Leu Glu Asn Gly Gly Thr His Arg Glu Lys Lys Glu 1925 1930 1935 agc acc cca tct aca gcc tcc ctc ccg tcc tat gac agt gta act aaa 5856 Ser Thr Pro Ser Thr Ala Ser Leu Pro Ser Tyr Asp Ser Val Thr Lys 1940 1945 1950 cct gaa aag gag aaa cag cag cgg gca gag gaa gga aga agg gaa aga 5904 Pro Glu Lys Glu Lys Gln Gln Arg Ala Glu Glu Gly Arg Arg Glu Arg 1955 1960 1965 gcc aaa aga caa aaa gag gtc aga gaa tcc aag tgt tagaggagalu 5950 Ala Lys Val Arg Glu Ser Lys Cys 1970 1975 1980 caaaaattca gtattataca gatctaaaac tcgcaagtga aagattgttt acaaacttcc 6010 tgaatattat caatgcagaa cagctgtgga gactctaacc tgaagatcta taccaaacgt 6070 cgtctgctta ccacgtaaca cagctgcatc ttgagcagtg acctccaagg gcaaagtacc 6130 ccgctcccta gacttacaga ttttctaatg cttgggcagg tggttactgc atgttccaca 6190 tcagtcaatg caacttagga caaaactaac cagatacaga aacagaagag aggctgccgg 6250 gaccagcata tttccgttgc agccaaatgg attttatttt ttcattttat tgattctcag 6310 aagcagaaag catcacttta aaagttcgtt tgttcatgca aactatattt gcattcttac 6370 attagttaag ctaagcagca aaaagaaaac acacacacac a ctcacattt agcccatgtc 6430 atttaattgt cagtttcttt gacataaagc gcatcttctc cacatgggct tcacgtggtt 6490 tggagatggg tgggggaaaa caatcaggtt tcttcaggct gaggaggact tgctcaggcc 6550 gattccaaac attgtgctcg ttcaatgcgt agaaatgatt tgcatgatgg catgccgtga 6610 tcagaagtca tgcatgagat ccatacacca caggacacta ctaatctagt cccttgcact 6670 gggtcagcct ttggacagga cccagccctg caccgttcac tgtatttgga gaaaatggta 6730 agagttccat accggctaca attctttgag ttcttaaaag tccttcatac accttctggg 6790 tagggaaaca accaactaat tgactaacac caccaacaac aaaaaacaaa cccaatccaa 6850 caagcagatg gatccgttgc gtgtatatgt ttaacagaca tctccaacat acagccattg 6910 ttgcacattt tgcaagatga actatttaat gctgctctgt gtccagtaca tgggggagac 6970 tttgatccca aatggaaataaataaataaataaataaataaataaataaataaag
【図１】ＳＣＮ８ＡのＰＣＲ増幅に用いたプライマーセ
ットと、これらのクライマー合成の基になった既知配列
を示す模式図である。FIG. 1 is a schematic diagram showing primer sets used for PCR amplification of SCN8A and known sequences from which these climbers are synthesized.
【図２】ＳＣＮ８ｃＤＮＡの構造と、ＲＴ−ＰＣＲおよ
びＲＡＣＥにより得られたクローンとの関係を示す模式
図である。FIG. 2 is a schematic diagram showing the relationship between the structure of SCN8 cDNA and clones obtained by RT-PCR and RACE.
【図３】ＳＣＮ８Ａ完全長ｃＤＮＡクローンの模式図で
ある。FIG. 3 is a schematic diagram of an SCN8A full-length cDNA clone.
───────────────────────────────────────────────────── フロントページの続き Ｆターム(参考） 4B024 AA01 BA80 CA04 DA02 DA06 EA04 GA11 HA01 HA12 4B064 AG27 CA20 CC24 DA13 4H045 AA11 AA20 CA40 DA76 DA86 EA21 FA72 FA74 GA06 GA10 GA23 GA25 GA26 ──────────────────────────────────────────────────続 き Continued on the front page F term (reference) 4B024 AA01 BA80 CA04 DA02 DA06 EA04 GA11 HA01 HA12 4B064 AG27 CA20 CC24 DA13 4H045 AA11 AA20 CA40 DA76 DA86 EA21 FA72 FA74 GA06 GA10 GA23 GA25 GA26
Claims (4)
リウムチャンネルＳＣＮ８。1. A sodium channel SCN8 having the amino acid sequence of SEQ ID NO: 1.
８Ａをコードするヒト遺伝子。2. The sodium channel SCN of claim 1.
Human gene encoding 8A.
て、配列番号１の塩基配列を有するｃＤＮＡ。3. The cDNA of the human gene according to claim 2, which has the nucleotide sequence of SEQ ID NO: 1.
ルＳＣＡ11Ａに対する抗体。4. An antibody against the sodium channel SCA11A of claim 1 or 2.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP11004645A JP2000201684A (en) | 1999-01-11 | 1999-01-11 | Sodium channel scn8 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP11004645A JP2000201684A (en) | 1999-01-11 | 1999-01-11 | Sodium channel scn8 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JP2000201684A true JP2000201684A (en) | 2000-07-25 |
Family
ID=11589714
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP11004645A Pending JP2000201684A (en) | 1999-01-11 | 1999-01-11 | Sodium channel scn8 |
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| Country | Link |
|---|---|
| JP (1) | JP2000201684A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2001090355A1 (en) * | 2000-05-23 | 2001-11-29 | Japan Science And Technology Corporation | Human sodium channel scn12a and scn8a |
-
1999
- 1999-01-11 JP JP11004645A patent/JP2000201684A/en active Pending
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2001090355A1 (en) * | 2000-05-23 | 2001-11-29 | Japan Science And Technology Corporation | Human sodium channel scn12a and scn8a |
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