Gating Kinetics of Single Large-Conductance Ca²⁺-Activated K⁺ Channels in High Ca²⁺ Suggest a Two-Tiered Allosteric Gating Mechanism^✪

The Ca²⁺-dependent gating mechanism of large-conductance calcium-activated K⁺ (BK) channels from cultured rat skeletal muscle was examined from low (4 μM) to high (1,024 μM) intracellular concentrations of calcium (Ca²⁺_i) using single-channel recording. Open probability (P_o) increased with increasing Ca²⁺_i (K_0.5 11.2 ± 0.3 μM at +30 mV, Hill coefficient of 3.5 ± 0.3), reaching a maximum of ∼0.97 for Ca²⁺_i ∼ 100 μM. Increasing Ca²⁺_i further to 1,024 μM had little additional effect on either P_o or the single-channel kinetics. The channels gated among at least three to four open and four to five closed states at high levels of Ca²⁺_i (>100 μM), compared with three to four open and five to seven closed states at lower Ca²⁺_i. The ability of kinetic schemes to account for the single-channel kinetics was examined with simultaneous maximum likelihood fitting of two-dimensional (2-D) dwell-time distributions obtained from low to high Ca²⁺_i. Kinetic schemes drawn from the 10-state Monod-Wyman-Changeux model could not describe the dwell-time distributions from low to high Ca²⁺_i. Kinetic schemes drawn from Eigen's general model for a ligand-activated tetrameric protein could approximate the dwell-time distributions but not the dependency (correlations) between adjacent intervals at high Ca²⁺_i. However, models drawn from a general 50 state two-tiered scheme, in which there were 25 closed states on the upper tier and 25 open states on the lower tier, could approximate both the dwell-time distributions and the dependency from low to high Ca²⁺_i. In the two-tiered model, the BK channel can open directly from each closed state, and a minimum of five open and five closed states are available for gating at any given Ca²⁺_i. A model that assumed that the apparent Ca²⁺-binding steps can reach a maximum rate at high Ca²⁺_i could also approximate the gating from low to high Ca²⁺_i. The considered models can serve as working hypotheses for the gating of BK channels.