Ludewig U, Pusch M, Jentsch T J
Center for Molecular Neurobiology, Hamburg University, Germany.
Biophys J. 1997 Aug;73(2):789-97. doi: 10.1016/S0006-3495(97)78111-6.
The Cl- channel from the Torpedo electric organ, CLC-0, is the prototype of a large gene family of Cl- channels. At the single-channel level, CLC-0 shows a "double-barreled" behavior. Recently it was shown that CLC-0 is a dimer, and it was suggested that each subunit forms a single pore. The two protopores are gated individually by a fast voltage and anion-dependent gating mechanism. A slower common gating mechanism operates on both pores simultaneously. Previously, wild-type/mutant heteromeric channels had been constructed that display a large wild-type pore and small mutant pore. Here we use patch-clamp recording of single wild-type and mutant CLC-0 channels to investigate in detail the dependence of the gating of one protopore on the physically attached neighboring pore. No difference in rate constants of opening and closing of protopores could be found comparing homomeric wild-type and heteromeric wild-type/mutant channels. In addition, detailed kinetic analysis reveals that gating of single subunits is not correlated with the gating of the neighboring subunit. The results are consistent with the view that permeation and fast gating of individual pores are fully independent of the neighboring pore. Because the two subunits are associated in a common protein complex, opening and closing transitions of individual pores are probably due to only small conformational changes in each pore. In addition to the fast and slow gating mechanisms known previously for CLC-0, in the course of this study we occasionally observed an additional gating process that led to relatively long closures of single pores.
来自电鳐电器官的氯离子通道CLC-0是氯离子通道一个大基因家族的原型。在单通道水平上，CLC-0表现出“双筒”行为。最近研究表明CLC-0是一种二聚体，并且有人提出每个亚基形成一个单一的孔道。两个原孔道通过一种快速的电压和阴离子依赖性门控机制独立门控。一种较慢的共同门控机制同时作用于两个孔道。此前，已经构建了野生型/突变体异源通道，其显示出一个大的野生型孔道和一个小的突变体孔道。在这里，我们使用膜片钳记录单个野生型和突变体CLC-0通道，以详细研究一个原孔道的门控对物理连接的相邻孔道的依赖性。比较同型野生型和异源野生型/突变体通道，未发现原孔道开放和关闭速率常数的差异。此外，详细的动力学分析表明，单个亚基的门控与相邻亚基的门控不相关。这些结果与单个孔道的通透和快速门控完全独立于相邻孔道的观点一致。由于两个亚基存在于一个共同的蛋白质复合物中，单个孔道的开放和关闭转变可能仅归因于每个孔道中较小的构象变化。除了先前已知的CLC- 的快速和慢速门控机制外，在本研究过程中，我们偶尔还观察到另一种门控过程，该过程导致单个孔道相对较长时间的关闭。
