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 mu M) to high (1,024 mu M) intracellular concentrations of calcium (Ca-i(2+),) using single-channel recording. Open probability (P-o) increased with increasing Ca-i(2+) (K-0.5 11.2 +/- 0.3 mu M at +30 mV, Hill coefficient of 3.5 +/- 0.3), reaching a maximum of similar to 0.97 for Ca-i(2+) similar to 100 mu M. Increasing Ca-i(2+) further to 1,024 mu M 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 Ca-i(2+) (>100 mu M), compared with three to four open and five to seven closed stares at lower Ca-i(2+). 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 fr om low to high Ca-i(2+). Kinetic schemes drawn from the 10-state Monod-Wyman-Changeux model could not describe the dwell-time distributions from low to high Ca-i(2+). 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 Ca-i(2+). 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 Ca-i(2+). 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 Ca-i(2+). A model that assumed that the apparent Ca2+-binding steps can reach a maximum rate at high Ca-i(2+) could also approximate the gating from low to high Ca-i(2+). The considered models can serve as working hypotheses for the gating of BK channels.