Horrigan F T, Cui J, Aldrich R W
Department of Molecular and Cellular Physiology, Howard Hughes Medical Institute, Stanford University School of Medicine, Stanford, California 94305, USA.
J Gen Physiol. 1999 Aug;114(2):277-304. doi: 10.1085/jgp.114.2.277.
Activation of large conductance Ca(2+)-activated K(+) channels is controlled by both cytoplasmic Ca(2+) and membrane potential. To study the mechanism of voltage-dependent gating, we examined mSlo Ca(2+)-activated K(+) currents in excised macropatches from Xenopus oocytes in the virtual absence of Ca(2+) (<1 nM). In response to a voltage step, I(K) activates with an exponential time course, following a brief delay. The delay suggests that rapid transitions precede channel opening. The later exponential time course suggests that activation also involves a slower rate-limiting step. However, the time constant of I(K) relaxation [tau(I(K))] exhibits a complex voltage dependence that is inconsistent with models that contain a single rate limiting step. tau(I(K)) increases weakly with voltage from -500 to -20 mV, with an equivalent charge (z) of only 0.14 e, and displays a stronger voltage dependence from +30 to +140 mV (z = 0.49 e), which then decreases from +180 to +240 mV (z = -0.29 e). Similarly, the steady state G(K)-V relationship exhibits a maximum voltage dependence (z = 2 e) from 0 to +100 mV, and is weakly voltage dependent (z congruent with 0.4 e) at more negative voltages, where P(o) = 10(-5)-10(-6). These results can be understood in terms of a gating scheme where a central transition between a closed and an open conformation is allosterically regulated by the state of four independent and identical voltage sensors. In the absence of Ca(2+), this allosteric mechanism results in a gating scheme with five closed (C) and five open (O) states, where the majority of the channel's voltage dependence results from rapid C-C and O-O transitions, whereas the C-O transitions are rate limiting and weakly voltage dependent. These conclusions not only provide a framework for interpreting studies of large conductance Ca(2+)-activated K(+) channel voltage gating, but also have important implications for understanding the mechanism of Ca(2+) sensitivity.
大电导钙激活钾通道的激活受细胞质钙和膜电位的共同控制。为了研究电压依赖性门控机制，我们在几乎不存在钙（<1 nM）的情况下，检测了非洲爪蟾卵母细胞切除的大膜片中的mSlo钙激活钾电流。响应电压阶跃时，I(K)在短暂延迟后以指数时间进程激活。该延迟表明通道开放之前存在快速转变。随后的指数时间进程表明激活还涉及一个较慢的限速步骤。然而，I(K)松弛的时间常数[tau(I(K))]表现出复杂的电压依赖性，这与包含单个限速步骤的模型不一致。tau(I(K))在电压从-500到-20 mV时随电压微弱增加，等效电荷（z）仅为0.14 e，而在+30到+140 mV时表现出更强的电压依赖性（z = 0.49 e），然后在+180到+240 mV时降低（z = -0.29 e）。同样，稳态G(K)-V关系在0到+100 mV时表现出最大电压依赖性（z = 2 e），在更负的电压下电压依赖性较弱（z约为0.4 e），此时P(o)=10(-5)-10(-6)。这些结果可以用一种门控机制来解释，即关闭和开放构象之间的中心转变由四个独立且相同的电压传感器的状态变构调节。在没有钙的情况下，这种变构机制导致一种具有五个关闭（C）和五个开放（O）状态的门控机制，其中通道的大部分电压依赖性来自快速的C-C和O-O转变，而C-O转变是限速的且电压依赖性较弱。这些结论不仅为解释大电导钙激活钾通道电压门控的研究提供了一个框架，而且对理解钙敏感性机制具有重要意义。
