WO2001096552A1

WO2001096552A1 - Sodium channels scn1a and scn3a

Info

Publication number: WO2001096552A1
Application number: PCT/JP2001/004956
Authority: WO
Inventors: Ichiro Kanazawa; Jun Goto; Seon-Yong Jeong
Original assignee: Japan Science And Technology Corporation
Priority date: 2000-06-13
Filing date: 2001-06-12
Publication date: 2001-12-20

Abstract

A human sodium channel SCN1A having the amino acid sequence represented by SEQ ID NO:2; a human gene encoding this protein molecule; and SCN1A cDNA having the base sequence represented by SEQ ID NO:1. Another human sodium channel SCN3A having the amino acid sequence represented by SEQ ID NO:4; a human gene encoding this protein molecule; and SCN3A cDNA. These substances largely contribute to the clarification of a physiological mechanism in which excitant cells participate, the specification of the causes of various human diseases, and the development of novel remedies.

Description

Description Natural channel SCN1A and SCN3A

TECHNICAL FIELD The invention of this application relates to novel α-subunits SCN1A and SCN3A of the human nervous system, genes and cDNAs thereof, and antibodies against SCN1A and SCN3A, respectively.

BACKGROUND ART Membrane potential-dependent sodium channels are membrane protein molecules 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, the action potential generated in the cell body propagates through the axon, thereby transmitting information as a function of the nerve cell. Sodium channels are protein molecules that, along with potassium channels, are responsible for the initiation and propagation of action potentials. The membrane potential-dependent sodium channel is composed of multiple subunits, the main function of which is provided in the a subunit. To date, the existence of multiple α-subunit genes has been clarified in mammals, and in humans, at least SCN1A (2q24), SCN2A (2q23), SCN3A (2q24-q31), and SCN4A (17q23) .1-q25.3), SCN5A (3p21), SCN6A (2q21-q23), SCN8A (12q13), SCN9A (2q24), SCN10A (3p22-q24) (Human chromosome loci in parentheses). However, all of these genes are completely cloned. Some genes are not reported, and only partial sequences are reported for some genes, or human homologous genes are identified from homologous sequences in mouse rats. In particular, for SCN1A and SCN3A, the full-length cDNAs have not yet been identified, and the entire proteins encoded by the genes are not known. In recent years, diseases caused by mutations in these genes have been identified in humans or animals, and it is clear that the α subunit of the sodium channel is not only a physiologically important molecule but also that the mutation causes disease. It has been to. For example, mutations in SCN4A have been shown to cause familial hyperkalemia-induced periodic limb paralysis, and mutations in SCN5A to cause long QT syndrome type 3. Although a corresponding disease in humans is not known, SCN8A has been cloned as a causative gene of mouse mutant motor endplate disease (med). Elucidation of the molecular biological entity and function of the membrane potential-dependent sodium channel is extremely important for understanding the physiological mechanism of humans and for identifying the direct cause of various diseases. . Furthermore, inferring from its physiological functions, substances that act on sodium channels can be drugs that regulate the function of cells in which they are expressed (eg, anesthetics, analgesics, muscle relaxants, etc.) It is also important as a target for the development of new therapeutic drugs. The inventors of the present application cloned genes for the purpose of confirming all sodium channels expressed in the human nervous system, and as a result, cloned the full-length cDNAs of human SCN1A and SCN3A. The inventors succeeded in elucidating the entire amino acid sequences of the proteins SCN1A and SCN3A encoded by this cDNA. This application describes a human sodium channel, SCN1A, discovered by the inventors. The task is to provide SCN3A and its cDNA specifically. Another object of the present application is to provide genes encoding SCN1A and SCN3A, respectively, and antibodies against SCN1A and SCN3A, respectively.

DISCLOSURE OF THE INVENTION The invention of this application provides a sodium channel SCN1A having the amino acid sequence of SEQ ID NO: 2 and a sodium channel SCN3A having the amino acid sequence of SEQ ID NO: 4. Also, the invention of this application is a human gene encoding the sodium channel SCN1A, which is present on the long arm of human chromosome 2 (2q24), and a cDNA of this gene, which has the nucleotide sequence of SEQ ID NO: 1. Provide cDNA. Furthermore, the invention of this application relates to a human gene encoding the above-mentioned sodium channel SCN3A, which is present on the long arm of human chromosome 2 (2q24-31), and a cDNA of this gene, which has the nucleotide sequence of SEQ ID NO: 3. Provide cDNA having Further, this application also provides an antibody against each of the aforementioned sodium channels SCN1A and SCN3A.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a schematic diagram showing primer sets used for PCR amplification of SCN1A and SCN3A, and known sequences from which these primers were synthesized. FIG. 2 is a schematic diagram showing the relationship between the structure of SCN1A cDNA and clones obtained by RT-PCR and RACE. FIG. 3 is a schematic diagram showing the relationship between the structure of SCN3A cDNA and clones obtained by RT-PCR and RACE. FIG. 4 shows the results of RT-PCR in which the expression of SCN1A mRNA in human nervous system tissues was examined. FIG. 5 shows the results of RT-PCR in which the expression of SCN3A mRNA in human nervous system tissues was examined.

BEST MODE FOR CARRYING OUT THE INVENTION The sodium channel SCN1A of the present invention is a transcript expressed from the cDNA whose nucleotide sequence is shown in SEQ ID NO: 1, and consists of the 1999 amino acids shown in SEQ ID NO: 2. . The sodium channel SCN3A of the present invention is a transcript expressed from the cDNA whose nucleotide sequence is shown in SEQ ID NO: 3, and is composed of 2000 amino acids shown in SEQ ID NO: 4. The SCN1A and SCN3A (hereinafter sometimes referred to as SCN1A; 3A) of the present invention can be obtained by a known method, that is, a method of isolating from human various organs, or a method based on the amino acid sequence provided by this application. The peptide can be obtained by a method of preparing a peptide by synthesis, or by a method of producing a recombinant DNA technique using cDNA provided by this application. For example, when SCN1A; 3A is obtained by recombinant DNA technology, RNA is prepared by in vitro transcription from a vector having a cDNA s translation region provided by the present invention, and this is used as an in vitro template for RNA preparation. B translation By doing so, SCN1A; 3A protein can be obtained. 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., SCN1A; 3A can be used in these transformants. It can be expressed in large quantities. When the SCN1A; 3A of the present invention is produced by in vitro translation, the translation region of the cDNA of the present invention is recombined into a vector having an RNA polymerase promoter. It may be added to an in vitro translation system such as a product or a wheat germ extract. Examples of the RNA polymerase promoter include T7, T3, and SP6. Examples of vectors containing these RNA polymerase promoters include pKA1, pCDM8, pT3 / T718, ρΤ7 / Τ319, pBluescript II and the like. When the SCNhA; 3A of the present invention is expressed in a microorganism such as Escherichia coli, an expression vector having an origin, a promoter, a ribosome binding site, a DNA cloning site, a terminator and the like capable of replicating in microorganisms may be used. An expression vector may be prepared by recombination of the translation region of the cDNA of the present invention, a host cell may be transformed with this expression vector, and the transformant may be cultured. At this time, if a start codon and a stop codon are added before and after the 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. By cutting this fusion protein with an appropriate protease, only SCN1A; 3A can be obtained. Examples of the expression vector for Escherichia coli include a pUC system, a pBluescript Ik pET expression system, and a pGEX expression system. When the SCN1A; 3A 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. Introduce into eukaryotic cells. Expression vectors include pKA1, pCDM8, pSVK3, pMSG, pSV, pBK-CMV, pBK-RSV, EBV Examples include pRS and pYES2. Eukaryotic cells include, but are not limited to, monkey kidney cells COS7, cultured mammalian cells such as Chinese hamster ovary cells, CHO, budding yeast, fission yeast, phyco-cells, and African egg cells. It is not done. In order to introduce the expression vector into eukaryotic cells, known methods such as an electroporation method, a calcium phosphate method, a ribosome method, and a DEAE dextran method can be used. After the protein is expressed in prokaryotic 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 denaturing agents such as urea or surfactants, sonication, enzymatic digestion, salt precipitation and solvent precipitation, dialysis, centrifugation, ultrafiltration, gel filtration, SDS-PAGE, isoelectric focusing , Ion exchange chromatography, hydrophobic chromatography, affinity chromatography, reverse phase chromatography and the like. The SCN1A; 3A of the present invention includes a peptide fragment (5 or more amino acid residues) containing any partial sequence in the amino acid sequence of SEQ ID NO: 2 or 4. These peptide fragments can be used as antigens for producing antibodies. The SCN1A; 3A of the present invention also includes a fusion protein with any other protein. The gene of the present invention is a gene that encodes the above-mentioned SCN1A and exists on the second long arm (2q24) of human chromosome. The gene of the present invention is a gene that encodes the above-mentioned SCN3A and is present on the second long arm (2q24-31) of the human chromosome. These genes can be isolated as DNA fragments from existing genomic libraries using, for example, the cDNA of the present invention or a partial sequence thereof as a probe. The cDNA of the present invention can be cloned, for example, from human cell-derived cDNA. cDNA synthesizes poly A + RNA extracted from human cells as a cycl type. Examples of the synthesis method include the Okayama-Ichi Berg method (ol. Cell. Biol. 2: 161-170, 1982) and the Gubler-Hoffman method (丄 Gene 25: 263-269, 1983), and the cabbing method (Gene 150: 243-250, 1994) can be used. A commercially available human cDNA library can also be used. To clone the cDNA of the present invention from the cDNA 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 hybridize by a known method. Screening using a dimension may be performed. In addition, an oligonucleotide that hybridizes to both ends of the desired cDNA fragment is synthesized, and using this as a primer, the cDNA of the present invention is prepared from mRNA isolated from human cells by the RT-PCR method. You can do that too. In general, polymorphisms due to individual differences are frequently observed in human genes. Accordingly, a cDNA of SEQ ID NO: 1 or 3 in which one or more nucleotides have been added, deleted, and substituted with other nucleotides or other nucleotides is also included in the cDNA of the present invention. Similarly, SCN1A having one or more amino acids added, deleted, and substituted by amino acids or other amino acids resulting from these base changes; SCN1A having the amino acid sequence of SEQ ID NO: 2 or 4 Alternatively, as long as it has SCN3A activity, it is included in the present invention. 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 3. It also includes a DNA fragment consisting of a sense strand and an antisense strand. These DNA fragments can be used as probes for gene diagnosis. The antibody of the present invention can be obtained as a polyclonal antibody or a monoclonal antibody by a known method using SCN1A or SCN3A itself or a partial peptide thereof as an antigen. Next, as an example, the background and characteristics of the acquisition of SCN1A and SCN3A of the present invention The following shows the results of the study on the above.

Example

(1) Purification of human autopsy brain mRNA

2 g of an autopsy brain slice from a non-neurological disease patient was completely crushed with a homogenizer together with 20 mL of TRIzol reagent (manufactured by Gibco-BRL) and incubated at 30 ° C for 5 minutes. Next, 4 mL of chloroform was added, stirred, and incubated at 30 ° C for 3 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. The polyA + RNA (mRNA) fraction was purified from total RNA using Dynabeads mRNA purification kit (DYNAL).

(2) RT-PCR

Single-stranded DNA was synthesized using supercrypt II reverse transcriptase (manufactured by Gibco-BRL), using the mRNA purified in (1) as type III and random primers. This single-stranded DNA is referred to as type I. As shown in Fig. 1, the homology is high between known sodium channel α subunits (human: Η and rat: R), and the well-conserved part of the sequence. A partial distribution sequence of a sodium channel containing a known sodium channel α-subunit was obtained by nested PCR using an oligonucleotide designed based on the above as a primer. One of the clones, 1615 bp, has a very high homology of 90% in the nucleotide sequence and 90% in the amino acid sequence of the portion corresponding to SCN1A cloned in the rat. It was confirmed that it was a partial sequence. The 1593 bp clone has a very high homology of 89% in base sequence and 97% in amino acid sequence corresponding to SCN3A cloned in the rat.Therefore, this clone contains human SCN3A cDNA. It was confirmed that it was a partial sequence. (3) RACE.

Primers were synthesized based on the base sequences of the partial sequences of the human SCN1A cDNA and SCN3A cDNA obtained in (2) above, and Marathon-ready cDNA (Clontech) was used as type III for 5'-RACE and 3'-RACE. Was performed to obtain 5 ′ and 3 ′ cDNAs. The 5 'end was obtained by PCR using primers designed based on the 5' end cDNA sequence of the rat. The relationship between the obtained cDNA fragments is as shown in FIGS.

(4) Subcloning

The respective cDNA fragments of SCN1A and SCN3A obtained by RT-PCR and RACE were subcloned into a plasmid vector pBleuscript IISK (+).

(5) Sequencing

The plasmid of the above (4) was propagated and purified, subjected to a sequencing reaction by a dye terminator and a die primer method, and the reaction product was analyzed by a fluorescent sequencer-377A (Perkin Elmer / ABI).

(6) Results and discussion

As shown in SEQ ID NO: 1, the cDNA of SCN1A is 8131 bp in total length, has a 5997 bp RF, and encodes a 1999 amino acid residue protein. In this SCN1A, four transmembrane domains common to the sodium channel α-subunit are identified. Table 1 shows the homology with the known sodium channel α subunit gene cDNA. The homology with the rat homologous gene was 97%. Also, among the known α-subunits, relatively high homology was observed between human SCN2A, rat Scn2a, and Scn3a. In order to investigate the expression site in detail, mRNA extracted from central nervous system tissues of humans As a result of RT-PCR using 錶 as a type II, SCN1A showed strong expression in all regions of the brain except for cerebral white matter, spinal cord and dorsal root ganglia (Fig. 4). Table 1 Human homology

(%) SCN2A SC 4A SCN5A SC 6A SCN8A SCN89A SCN1QA Scn1 Scn2a Scn3a

SCN1A '89 68 61 50 80 75 56 97 87 83

As shown in SEQ ID NO: 3, the cDNA of SCN3A is 9123 bp in total length, has a SOOObp ORF, and encodes a protein of 2000 amino acid residues. In this SCN3A, four transmembrane domains common to the sodium channel α-subunit are identified. Table 2 shows the homology with the known sodium channel 0: subunit gene cDNA. The homology with the rat homologous gene was 97%. Also, among the known α-subunits, relatively high homology was observed between human SCN2A, rat Scn1a, and Scn2a. In addition, in order to examine the detailed expression site, RT-PCR was performed using mRNA extracted from the central nervous system tissues of humans as a result of RT-PCR. Expression was also observed in the dorsal root ganglion (Fig. 5). Table 2 Human homology

(%) SC 2A SCN ^ SCN5A SCN6A SCN8A SCN89A SCN1QA Scn1 Scn2a Scn3a

SCN3A 81 70 62 50 76 75 57 84 86 97 INDUSTRIAL APPLICABILITY As described in detail above, the present application provides human sodium channel α-subunits SCN1A and SCN3A, and cDNAs encoding them. 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.

Claims

The scope of the claims

1. A sodium channel SCN1A having the amino acid sequence of SEQ ID NO: 2.

2. A sodium channel SCN3A having the amino acid sequence of SEQ ID NO: 4.

3. A human gene encoding the sodium channel SCN1A of claim 1 and present on the long arm of human chromosome 2 (2q24).

4. A human gene encoding the sodium channel SCN3A of claim 2 and present on the long arm of human chromosome 2 (2q24-31).

5. A cDNA of the human gene according to claim 3, which has the nucleotide sequence of SEQ ID NO: 1.

6. A cDNA of the human gene according to claim 4, which has the nucleotide sequence of SEQ ID NO: 3.

7. An antibody against the sodium channel SCN1A of claim 1.

8. An antibody against the sodium channel SCN3A of claim 2.