From the Department of Physiology and Biophysics, University of Miami School of Medicine, Miami, Florida 33101-6430 abstract 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, qeff, of 2.3 0.6 eo). Estimates of qeff were little changed for intracellular Ca2(Ca2 i) ranging from 0.0003 to 1,024 M. Increasing Ca2 i from 0.03 to 1,024 M shifted the voltage for half maximal activation (V1/2) 175 mV in the hyperpolarizing direction. V1/2 was independent of Ca2 i for Ca2 i 0.03 M, 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 (qeff 0.5 eo) and an increase in the mean opening rate (qeff 1.8 eo), 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 (0 through 1,024 M), voltage (80 to 80 mV), and Po (10 4 to 0.96). In the model, the voltage dependence of the gating arises mainly from voltagedependent transitions between closed (CC) and open (OO) states, with less voltage dependence for transitions between open and closed states (CO), 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.