Structure and function of the tetrodotoxin-resistant sodium channel

抗河豚毒素钠通道的结构和功能

• 批准号: 02454146
• 负责人: NAKAZAWA Atsushi
• 金额: $ 3.01万
• 依托单位: Yamaguchi University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (B)
• 财政年份: 1990
• 资助国家: 日本
• 起止时间: 1990 至 1991
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-02454146/
• 关键词: Sodium channel; Tetrodotoxin; cDNA; Puffer fish; ナトリウムチヤネル; フグ

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.

已经阐明了钠通道的一级结构，其有助于神经系统中动作电位的产生。然而，河豚毒素的结合位点，通道阻滞剂仍然是未确定的。本研究以河豚鱼脑组织为材料，以寡核苷酸（dT）和随机序列DNA为引物，构建了河豚鱼钠通道的cDNA文库，并对河豚鱼钠通道进行了序列分析，以确定河豚毒素在钠通道上的结合位点。利用PCR扩增的cDNA片段，我们分离出9个cDNA克隆。两个克隆都不含全长（6 kb）cDNA，但通过将分离的克隆的所有序列组合在一起来覆盖通道的整个编码序列。河豚鱼脑中至少存在3种不同的钠通道序列，通过与其他来源的钠通道序列比较，发现河豚毒素抗性钠通道的4个重复序列中，第一个重复序列的第5 ~ 6段之间存在缺失。这个区域，IS 5-IS 6，被认为是从细胞膜突出到细胞外部，并且是碳水化合物修饰的目标。河豚毒素的敏感性似乎是由某些氨基酸残基的延伸，而不是由一个单一的氨基酸取代。

项目成果

M.Yamada: "Characterization of cDNA encoding tetrodotoxin-resistant sodium chennel from puffer fish"

M.Yamada：“河豚鱼抗河豚毒素钠盐 cDNA 的表征”

M. Yamada, M. Shahjahan, M. Nagaya, and A. Nakazawa: "Characterization of cDNA encoding tetrodotoxin-resistant sodium channel from puffer fish"

M. Yamada、M. Shahjahan、M. Nagaya 和 A. Nakazawa：“河豚鱼抗河豚毒素钠通道 cDNA 的表征”

H.Tanaka: "Isolation and characterization of the gene encoding bovine adenylate kinase isozyme 2" Gene. 93. 221-227 (1990)

H.Tanaka：“牛腺苷酸激酶同工酶 2 编码基因的分离和表征”基因。

M.Yamada: "Characterization of cDNA encoding tetrodotoxinーresistant sodium channel from puffer fish"

M.Yamada：“河豚鱼抗河豚毒素钠通道 cDNA 的表征”