Structure and function of the tetrodotoxin-resistant sodium channel

钠通道的初级结构，有助于神经系统动作电位的产生，已经被阐明。然而，通道阻滞剂河豚毒素的结合部位仍未确定。本课题通过对河豚抗河豚毒素通道的序列分析，以确定河豚毒素在钠通道上的结合部位，从河豚脑组织出发，以对偶(Dt)和随机序列DNA为引物，在lambdagtll载体上构建了两种类型的cDNA库。利用聚合酶链式反应(PCR)扩增出的DNA片段和从cDNA中提取的DNA片段，分离出9个克隆。两个克隆都不包含全长(6kb)的cDNA，但通过将分离克隆的所有序列组合在一起，覆盖了通道的整个编码序列。河豚脑中至少存在3个不同的钠通道序列，通过与其他来源的钠通道氨基酸序列比较，发现河豚抗河豚毒素钠通道的4个重复序列中，第1个重复序列的第5~6个片段之间存在缺失。这个区域，IS5-IS6，被认为是从细胞膜突出到细胞外的区域，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决定的，而不是由单一的氨基酸替代决定的。

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.