Revilla A, Castro C, Barrio L C
Unidad Neurología Experimental-C.S.I.C., Departamento de Investigación, Hospital "Ramón y Cajal," 28034 Madrid, Spain.
Biophys J. 1999 Sep;77(3):1374-83. doi: 10.1016/S0006-3495(99)76986-9.
Most gap junction channels are sensitive to the voltage difference between the two cellular interiors, termed the transjunctional voltage (V(j)). In several junctions, the conductance transitions induced by V(j) show more than one kinetic component. To elucidate the structural basis of the fast and slow components that characterize the V(j )dependence of connexin-32 (Cx32) and connexin-43 (Cx43) junctions, we created deletions of both connexins, where most of the carboxy-terminal (CT) domain was removed. The wild-type and "tailless" mutants were expressed in paired Xenopus oocytes, and the macroscopic gating properties were analyzed using the dual voltage clamp technique. Truncation of the CT domain of Cx32 and Cx43 abolished the fast mechanism of conductance transitions and induced novel gating properties largely attributable to the slow mechanism of gating. The formation of hybrid junctions comprising wild-type and truncated hemichannels allowed us to infer that the fast and slow components of gating reside in each hemichannel and that both gates close at a negative V(j) on the cytoplasmic side. Thus we conclude that the two kinetic components of V(j)-sensitive conductance are a result of the action of two different gating mechanisms. They constitute separate structures in the Cx32 and Cx43 molecules, the CT domain being an integral part of fast V(j) gating.
大多数间隙连接通道对两个细胞内部之间的电压差敏感，该电压差称为跨连接电压（V(j)）。在几个连接处，由V(j)诱导的电导转变表现出不止一个动力学成分。为了阐明表征连接蛋白32（Cx32）和连接蛋白43（Cx43）连接处V(j)依赖性的快速和慢速成分的结构基础，我们构建了两种连接蛋白的缺失体，其中大部分羧基末端（CT）结构域被去除。野生型和“无尾”突变体在成对的非洲爪蟾卵母细胞中表达，并使用双电压钳技术分析宏观门控特性。Cx32和Cx43的CT结构域的截短消除了电导转变的快速机制，并诱导了主要归因于慢速门控机制的新门控特性。包含野生型和截短半通道的杂合连接的形成使我们推断，门控的快速和慢速成分存在于每个半通道中，并且两个门在细胞质侧的负V(j)处关闭。因此我们得出结论，V(j)敏感电导的两个动力学成分是两种不同门控机制作用的结果。它们在Cx32和Cx43分子中构成独立的结构，CT结构域是快速V(j)门控的一个组成部分。
