Yusef Yamil R, Zúñiga Leandro, Catalán Marcelo, Niemeyer María Isabel, Cid L Pablo, Sepúlveda Francisco V
Centro de Estudios Cientificos (CECS), Av. Arturo Prat 514, Valdivia, Chile.
J Physiol. 2006 Apr 1;572(Pt 1):173-81. doi: 10.1113/jphysiol.2005.102392. Epub 2006 Feb 9.
Functional and structural studies demonstrate that Cl(-) channels of the ClC family have a dimeric double-barrelled structure, with each monomer contributing an identical pore. Studies with ClC-0, the prototype ClC channel, show the presence of independent mechanisms gating the individual pores or both pores simultaneously. A single-point mutation in the CBS-2 domain of ClC-0 has been shown to abolish slow gating. We have taken advantage of the high conservation of CBS domains in ClC channels to test for the presence of a slow gate in ClC-2 by reproducing this mutation (H811A). ClC-2-H811A showed faster opening kinetics and opened at more positive potentials than ClC-2. There was no difference in Cl(-) dependence. Additional neutralization of a putative pore gate glutamate side chain (E207V) abolished all gating. Resolving slow and fast gating relaxations, however, revealed that the H811A mutation affected both fast and slow gating processes in ClC-2. This suggests that slow and fast gating in ClC-2 are coupled, perhaps with slow gating contributing to the operation of the pore E207 as a protopore gate.
功能和结构研究表明，ClC家族的氯离子通道具有二聚体双桶结构，每个单体都有一个相同的孔道。对原型ClC通道ClC-0的研究表明，存在独立的机制分别控制单个孔道或同时控制两个孔道。已证明ClC-0的CBS-2结构域中的单点突变会消除慢门控。我们利用ClC通道中CBS结构域的高度保守性，通过重现此突变（H811A）来测试ClC-2中是否存在慢门控。ClC-2-H811A表现出更快的开放动力学，并且比ClC-2在更正的电位下开放。[Cl⁻]i依赖性没有差异。对假定的孔道门控谷氨酸侧链（E207V）的进一步中和消除了所有门控。然而，解析慢门控和快速门控弛豫发现，H811A突变影响了ClC-2中的快速和慢门控过程。这表明ClC-2中的慢门控和快速门控是耦合的，可能慢门控有助于孔道E207作为原孔道门控的运作。
