Structure and function of the tetrodotoxin-resistant sodium channel

在神经系统中，参与动作电位产生的钠离子通道的主要结构已被阐明。然而，河豚毒素的结合位点，一种通道阻滞剂仍未确定。本项目对具有河豚毒素抗性通道的河豚鱼的钠通道进行序列分析，旨在鉴定河豚毒素在钠通道上的结合位点。从河豚脑组织开始，以同源（dT）和随机序列DNA为引物，在lambdagtll载体上构建了两类cDNA文库。利用PCR得到的cDNA片段，分离出9个cDNA克隆。两个克隆均未包含全长cDNA (6kb)，但通过将分离克隆的所有序列组合在一起，覆盖了整个通道编码序列。在河豚的大脑中至少存在3种不同的钠通道序列。将推断的河豚鱼通道氨基酸序列与其他来源的通道序列进行比较，发现在河豚毒素钠通道的4个重复序列中，第一个重复序列的第5段和第6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，是碳水化合物修饰的目标。河豚毒素的敏感性似乎是由氨基酸残基的某些延伸决定的，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.