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Reconstruction of native K^+ channels by use of cloned K^+ channels and its application to the development of antiarrhythmic agents.

利用克隆的K^通道重建天然K^通道及其在抗心律失常药物开发中的应用。

基本信息

批准号：
07557170
负责人：
ISHII Kuniaki
金额：
$ 7.74万
依托单位：
Yamagata University (1996-1997)Tohoku University (1995)
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (A)
财政年份：
1995
资助国家：
日本
起止时间：
1995 至 1996
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-07557170/
关键词：
K^+ channel clones antiarrhythmic drugs quinidine MS-551 verapamil Herg サブユニット内向き整流

项目摘要

We had reported that the class III antiarrhythmic drugs had no effects on the currents of cloned K^+ channels (Kv family). Lack of auxiliary subunit (S) which affects the sensitivity of K^+ channel clones to drugs was one of the possibilities. However, recently two (three) K^+ channel genes that are responsible for I_<kr> (Herg) and I_<ks> (KvLQT1+minK) have been identified. Mutation of these genes causes long QT syndrome. Since, I_<kr> is a target for typical class III drugs, we had decided to study Herg channel not Kv channels. Effects of quinidine (class I), MS-551 (class III) and verapamil (class IV) on the currents flowing through Herg channel were investigated using a X enopus oocyte expression system. The three drugs blocked Herg currents in a concentration-dependent manner. The concentration required to reduce tail-current by 50% (IC50) was about 3.2muM for quinidine, 8.8muM for MS-551 and 7.3muM for verapamil at 0 mV.IC50 for quinidine and verapamil did not vary significantly with test potential, while that for MS-551 seemed to be smaller with larger depolarizing test pulse. When MS-551 or verapamil was applied, repetitive pulsing to 0 mV caused a slight cumulative decrease of Herg currents. When quinidine was applied, a cumulative decrease in Herg currents was not evident with repetitive pulsing. Quinidine is known to act as open channel blocker of native K^+ currents. Therefore it might be possible that quinidine blocks open Herg channel very rapidly. Recovery of Herg currents from the block by the drugs were also investigated. After a 10-min washout, Herg currents recovered 80% from the block by MS-551 (30muM), but hardly recovered from the block by verapamil (30muM). Several point mutants were constructed to study the binding sites of the antiarrhythmic drugs. Preliminary results indicate that the sixth transmembrane is probably involved in the binding of the drugs.

我们曾报道过III类抗心律失常药物对克隆的K~+通道(Kv家族)电流无影响。缺乏影响K~+通道克隆对药物敏感性的辅助亚基(S)是可能的原因之一。然而，最近发现了两个与I_&lt；Kr&gt；(HERG)和I_&lt；ks&gt；(KvLQT1+mink)相关的K^+通道基因。这些基因的突变会导致长QT综合征。由于I_&lt；Kr&gt；是典型的III类药物的靶点，我们决定研究Herg通道而不是Kv通道。用X enopus卵母细胞表达系统研究了奎尼丁(I类)、MS-551(III类)和维拉帕米(IV类)对HERG通道电流的影响。这三种药物以浓度依赖的方式阻断Herg电流。在0 mV时，奎尼丁、MS-551和维拉帕米的IC50分别约为3.2、8.8和7.3微米。奎尼丁和维拉帕米的IC50随测试电压的变化不大，而MS-551的IC50随去极化测试脉冲的增大而减小。当使用MS-551或维拉帕米时，重复脉冲至0 mV时，HERG电流略有累积减小。当应用奎尼丁时，重复脉冲对Herg电流的累积减少并不明显。奎尼丁被认为是天然K~+电流的开放通道阻滞剂。因此，奎尼丁可能会非常迅速地阻断开放的HERG通道。研究了药物对阻断后Herg电流的恢复作用。洗涤10min后，MS-551(30微米)可使HERG电流恢复80%，而维拉帕米(30微米)则几乎不能使其恢复。构建了几个点突变株来研究抗心律失常药物的结合部位。初步结果表明，第六跨膜可能参与药物的结合。