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

点击翻译按钮获取中文摘要

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.