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。

• 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; K^+ channel clones; antiarrhythmic drugs; quinidine; MS-551; verapamil; Herg; サブユニット; 内向き整流

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.

我们报道说，III类抗心律失常药物对克隆K^+通道的电流没有影响。缺乏影响K^+通道克隆对药物敏感性的辅助亚基是可能性之一。但是，最近已经确定了负责I_ <kr>（HERG）和I_ <ks>（KVLQT1+ Mink）的两个（三）K^+通道基因。这些基因的突变导致QT综合征长。由于I_ <kr>是典型的III类药物的目标，因此我们决定研究HERG通道而不是KV通道。使用X Enopus卵母细胞表达系统研究了奎尼丁（I类），MS-551（III类）和Verapamil（IV类）对流过HERG通道的电流的影响。这三种药物以浓度依赖性方式阻止了HERG电流。奎尼丁降低尾电流所需的浓度约为3.2mum，MS-551的8.8MUM和quinidine和Verapamil的Verapamil在0 mV.IC50的7.3mum和7.3mum在测试势方面并没有很大变化，而对于MS-551的较小，具有较小的测试脉冲的较小。当应用MS-551或Verapamil时，重复脉冲至0 mV导致HERG电流略有累积降低。当应用奎尼丁时，随着重复的脉动，HERG电流的累积减少并不明显。奎尼丁被称为天然K^+电流的开放通道阻滞剂。因此，奎尼丁阻滞可能会非常迅速地打开HERG通道。还研究了通过药物从块中恢复HERG电流。经过10分钟的冲洗后，HERG电流通过MS-551（30MUM）从块中恢复了80％，但几乎没有通过Verapamil（30MUM）从块中回收。构建了几个点突变体以研究抗心律失常药物的结合位点。初步结果表明，第六跨膜可能与药物的结合有关。