Bukauskas F F, Weingart R
Department of Physiology, University of Bern, Switzerland.
Biophys J. 1994 Aug;67(2):613-25. doi: 10.1016/S0006-3495(94)80521-1.
De novo formation of cell pairs was used to examine the gating properties of single gap junction channels. Two separate cells of an insect cell line (clone C6/36, derived from the mosquito Aedes albopictus) were pushed against each other to provoke formation of gap junction channels. A dual voltage-clamp method was used to control the voltage gradient between the cells (Vj) and measure the intercellular current (Ij). The first sign of channel activity was apparent 4.7 min after cell contact. Steady-state coupling reached after 30 min revealed a conductance of 8.7 nS. Channel formation involved no leak between the intra- and extracellular space. The first opening of a newly formed channel was slow (25-28 ms). Each preparation passed through a phase with only one operational gap junction channel. This period was exploited to examine the single channel properties. We found that single channels exhibit several conductance states with different conductances gamma j; a fully open state (gamma j(main state)), several substates (gamma j(substates)), a residual state (gamma j(residual)) and a closed state (gamma j(closed)). The gamma j(main state) was 375 pS, and gamma j(residual) ranged from 30 to 90 pS. The transitions between adjacent substates were 1/7-1/4 of gamma j(main state). Vj had no effect on gamma j(main state), but slightly affected gamma j (residual). The lj transitions involving gamma j(closed) were slow (15-60 ms), whereas those not involving gamma j(closed) were fast (< 2 ms). An increase in Vj led to a decrease in open channel probability. Depolarization of the membrane potential (Vm) increased the incidence of slow transitions leading to gamma j(closed). We conclude that insect gap junctions possess two gates, a fast gate controlled by Vj and giving rise to gamma j(substates) and gamma j(residual), and a slow gate sensitive to Vm and able to close the channel completely.
利用细胞对的从头形成来研究单个缝隙连接通道的门控特性。将昆虫细胞系（克隆C6/36，源自白纹伊蚊）的两个独立细胞相互挤压，以促使缝隙连接通道的形成。采用双电压钳方法来控制细胞间的电压梯度（Vj）并测量细胞间电流（Ij）。细胞接触后4.7分钟，通道活动的第一个迹象明显出现。30分钟后达到的稳态耦合显示电导为8.7 nS。通道形成过程中细胞内和细胞外空间之间没有泄漏。新形成通道的首次开放较慢（25 - 28毫秒）。每个制备物都经历了只有一个功能性缝隙连接通道的阶段。利用这个时期来研究单通道特性。我们发现单通道表现出几种具有不同电导γj的电导状态；一个完全开放状态（γj（主状态））、几个亚状态（γj（亚状态））、一个残余状态（γj（残余））和一个关闭状态（γj（关闭））。γj（主状态）为375 pS，γj（残余）范围为30至90 pS。相邻亚状态之间的转变为γj（主状态）的1/7 - 1/4。Vj对γj（主状态）没有影响，但对γj（残余）有轻微影响。涉及γj（关闭）的Ij转变较慢（15 - 60毫秒），而不涉及γj（关闭）的转变较快（<2毫秒）。Vj的增加导致开放通道概率降低。膜电位（Vm）的去极化增加了导致γj（关闭）的缓慢转变的发生率。我们得出结论，昆虫缝隙连接具有两个门控，一个由Vj控制的快速门控，产生γj（亚状态）和γj（残余），以及一个对Vm敏感且能够完全关闭通道的缓慢门控。
