Rothberg B S, Magleby K L
Department of Physiology and Biophysics, University of Miami School of Medicine, Miami, Florida 33101-6430, USA.
J Gen Physiol. 2000 Jul 1;116(1):75-99. doi: 10.1085/jgp.116.1.75.
The voltage- and Ca2+-dependent gating mechanism of large-conductance Ca2+-activated K+ (BK) channels from cultured rat skeletal muscle was studied using single-channel analysis. Channel open probability (Po) increased with depolarization, as determined by limiting slope measurements (11 mV per e-fold change in Po; effective gating charge, q(eff), of 2.3 +/- 0.6 e(o)). Estimates of q(eff) were little changed for intracellular Ca2+ (Ca2+(i)) ranging from 0.0003 to 1,024 microM. Increasing Ca2+(i) from 0.03 to 1,024 microM shifted the voltage for half maximal activation (V(1/2)) 175 mV in the hyperpolarizing direction. V(1/2) was independent of Ca2+(i) for Ca2+(i) < or = 0.03 microM, indicating that the channel can be activated in the absence of Ca2+(i). Open and closed dwell-time distributions for data obtained at different Ca2+(i) and voltage, but at the same Po, were different, indicating that the major action of voltage is not through concentrating Ca2+ at the binding sites. The voltage dependence of Po arose from a decrease in the mean closing rate with depolarization (q(eff) = -0.5 e(o)) and an increase in the mean opening rate (q(eff) = 1.8 e(o)), consistent with voltage-dependent steps in both the activation and deactivation pathways. A 50-state two-tiered model with separate voltage- and Ca2+-dependent steps was consistent with the major features of the voltage and Ca2+ dependence of the single-channel kinetics over wide ranges of Ca2+(i) (approximately 0 through 1,024 microM), voltage (+80 to -80 mV), and Po (10(-4) to 0.96). In the model, the voltage dependence of the gating arises mainly from voltage-dependent transitions between closed (C-C) and open (O-O) states, with less voltage dependence for transitions between open and closed states (C-O), and with no voltage dependence for Ca2+-binding and unbinding. The two-tiered model can serve as a working hypothesis for the Ca2+- and voltage-dependent gating of the BK channel.
采用单通道分析方法，研究了培养的大鼠骨骼肌中大电导钙激活钾（BK）通道的电压和钙依赖性门控机制。通过极限斜率测量确定，通道开放概率（Po）随去极化而增加（Po每增加e倍变化，斜率为11 mV；有效门控电荷q(eff)为2.3±0.6 e(o)）。对于细胞内钙（Ca2+(i)）浓度范围从0.0003到1024 μM，q(eff)的估计变化不大。将Ca2+(i)从0.03增加到1024 μM，使半数最大激活电压（V(1/2)）向超极化方向移动175 mV。当Ca2+(i)≤0.03 μM时，V(1/2)与Ca2+(i)无关，表明该通道在无Ca2+(i)时也可被激活。在不同Ca2+(i)和电压下但Po相同的数据中，开放和关闭驻留时间分布不同，这表明电压的主要作用不是通过在结合位点聚集Ca2+。Po的电压依赖性源于去极化时平均关闭速率降低（q(eff)= -0.5 e(o)）和平均开放速率增加（q(eff)= 1.8 e(o)），这与激活和失活途径中的电压依赖性步骤一致。一个具有独立电压和钙依赖性步骤的50状态两层模型与在广泛的Ca2+(i)（约0至1024 μM）、电压（+80至 -80 mV）和Po（10(-4)至0.96）范围内单通道动力学的电压和钙依赖性的主要特征一致。在该模型中，门控的电压依赖性主要源于关闭（C-C）和开放（O-O）状态之间的电压依赖性转变，开放和关闭状态（C-O）之间的电压依赖性较小，而Ca2+结合和解离无电压依赖性。该两层模型可作为BK通道钙和电压依赖性门控的一个工作假设。
