| Project/Area Number |
02454146
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| Research Category |
Grant-in-Aid for General Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Research Field |
General medical chemistry
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| Research Institution | Yamaguchi University |
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Principal Investigator |
NAKAZAWA Atsushi Yamaguchi Univ. Sch. Med. Professor, 医学部, 教授 (90025594)
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| Co-Investigator(Kenkyū-buntansha) |
YAMADA Mamoru Yamaguchi Univ. fac. Agr. Associate Prof., 農学部, 助教授 (30174741)
NOMA Takafumi Yamaguchi Univ. Sch. Med. Assistant Prof., 医学部, 講師 (40189428)
伴 隆志 山口大学, 医学部, 教授 (50101069)
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| Project Period (FY) |
1990 – 1991
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| Project Status |
Completed (Fiscal Year 1991)
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| Budget Amount *help |
¥4,700,000 (Direct Cost: ¥4,700,000)
Fiscal Year 1991: ¥1,500,000 (Direct Cost: ¥1,500,000)
Fiscal Year 1990: ¥3,200,000 (Direct Cost: ¥3,200,000)
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| Keywords | Sodium channel / Tetrodotoxin / cDNA / Puffer fish / ナトリウムチヤネル / フグ |
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| Research Abstract |
The primary structures of the sodium channel, which contributes to generation of the action potential in the nervous system, have been elucidated. However, the binding site of tetrodotoxin, a channel blocker is still undetermined. This project concerns the sequence analysis of the sodium channel of the puffer fish which has a tetrodotoxin-resistant channel, aiming at the identification of the binding site of tetrodotoxin on the sodium channel.
Starting from the brain tissues of the puffer fish, we constructed in the lambdagtll vector two types of cDNA libraries with ologo- (dT) and random sequence DNA as primers. Using DNA fragments that were made by PCR and prepared from cDNA, we isolated 9 cDNA clones. Neither clone contained a full-length (6 kb) cDNA, but the entire coding sequence of the channel was covered by combining all sequences of the isolated clones together. At least 3 different sequences of the sodium channel existed in the puffer fish brain.
By comparison of the deduced amino acid sequences of the puffer fish channels with the sequences of the channels from other sources, deletions were recognized in the region between the segments 5 and 6 of the first repeat sequence among 4 repeats in the tetrodotoxin-resistant sodium channels. This region, IS5-IS6, is thought to protrude from the membrane to the outside of the cell and to be a target of carbohydrate modifications. The tetrodotoxin sensitivity seems to be determined by certain stretches of amino acid residues and not by a single amino-acid replacement.
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