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 microM) to high (1,024 microM) intracellular concentrations of calcium (Ca2+i) using single-channel recording. Open probability (Po) increased with increasing Ca2+i (K0. 5 11.2 +/- 0.3 microM at +30 mV, Hill coefficient of 3.5 +/- 0.3), reaching a maximum of approximately 0.97 for Ca2+i approximately 100 microM. Increasing Ca2+i further to 1,024 microM had little additional effect on either Po 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 microM), 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.
Rothberg
B S
BS
Department of Physiology and Biophysics, University of Miami School of Medicine, Miami, Florida 33101-6430, USA.
Magleby
K L
KL
D000818:Animals; D002118:Calcium / Q000494:pharmacology; D004553:Electric Conductivity; D015640:Ion Channel Gating / Q000502:physiology*; D007700:Kinetics; D051036:Large-Conductance Calcium-Activated Potassium Channels; D008024:Ligands; D008954:Models, Biological; D009994:Osmolar Concentration; D015221:Potassium Channels / Q000187:drug effects / Q000378:metabolism* / Q000502:physiology; D024681:Potassium Channels, Calcium-Activated; D051381:Rats
D051036:Large-Conductance Calcium-Activated Potassium Channels; D008024:Ligands; D015221:Potassium Channels; D024681:Potassium Channels, Calcium-Activated; D002118:Calcium