Melishchuk A, Loboda A, Armstrong C M
Department of Physiology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104, USA.
Biophys J. 1998 Oct;75(4):1828-35. doi: 10.1016/S0006-3495(98)77624-6.
In potassium-free solutions some types of K channels enter a long-lasting nonconducting or "defunct" state. It is known that Shaker K channels must open in K+-free solutions to become defunct. Gating current studies presented here indicate an abnormal conformation in the defunct state that restricts S4 movement and alters its kinetics. Thus an abnormality initiated in the P region spreads to the gating apparatus. We find that channels most readily become defunct on repolarization to an intermediate voltage, thus prolonging occupancy of one of the several intermediate closed states. The state dependence of becoming defunct was further dissected by using the gating mutant L382A. Simply closing this channel at 0 mV (reversing the last activation step) does not make the mutant channel defunct. Instead, it is necessary to move further left (more fully closed) in the activation sequence. This was confirmed with ShIR experiments showing that channels become defunct only if there is inward gating charge movement. Rapid transit through the intermediate states, achieved at very negative voltage, is relatively ineffective at making channels defunct. Several mutations that removed C-type inactivation also made the channels resistant to becoming defunct. Our results show that normal gating current cannot be stably recorded in the absence of K+.
在无钾溶液中，某些类型的钾通道会进入一种持久的非导通或“失效”状态。已知Shaker钾通道在无钾溶液中必须打开才能失效。本文所呈现的门控电流研究表明，失效状态下存在一种异常构象，这种构象会限制S4的移动并改变其动力学。因此，在P区域引发的异常会扩散到门控装置。我们发现，通道在复极化到中间电压时最容易失效，从而延长了几种中间关闭状态之一的占据时间。通过使用门控突变体L382A进一步剖析了失效的状态依赖性。简单地在0 mV时关闭该通道（逆转最后一个激活步骤）并不会使突变通道失效。相反，有必要在激活序列中进一步向左移动（更完全关闭）。这通过ShIR实验得到了证实，该实验表明只有当存在内向门控电荷移动时通道才会失效。在非常负的电压下实现的快速通过中间状态，在使通道失效方面相对无效。几个消除C型失活的突变也使通道对失效具有抗性。我们的结果表明，在没有钾离子的情况下无法稳定记录正常的门控电流。
