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Gating kinetics of single large-conductance Ca2+-activated K+ channels in high Ca2+ suggest a two-tiered allosteric gating mechanism.

Rothberg BS, Magleby KL - J. Gen. Physiol. (1999)

Bottom Line: 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 considered models can serve as working hypotheses for the gating of BK channels.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology and Biophysics, University of Miami School of Medicine, Miami, Florida 33101-6430, USA.

ABSTRACT
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.

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Mentions: For the gating of the channel, the states in the top row of Fig. 1 are assumed to represent closed states of the channel, and the states in the bottom row are assumed to represent open states. The conductance of the states in the middle three rows is less clear, but may be open, closed, or partially conducting (Cox et al. 1997a). Fig. 1 reduces to the 35-state model of Eigen 1968 if it is assumed that diagonal and adjacent subunits in the same conformation have identical properties. Because of the complexity of the 55- and 35-state schemes, Fig. 1 is often reduced further to the 25-state model described by Fig. 2 (Fersht 1985).


Gating kinetics of single large-conductance Ca2+-activated K+ channels in high Ca2+ suggest a two-tiered allosteric gating mechanism.

Rothberg BS, Magleby KL - J. Gen. Physiol. (1999)

© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2229641&req=5

Mentions: For the gating of the channel, the states in the top row of Fig. 1 are assumed to represent closed states of the channel, and the states in the bottom row are assumed to represent open states. The conductance of the states in the middle three rows is less clear, but may be open, closed, or partially conducting (Cox et al. 1997a). Fig. 1 reduces to the 35-state model of Eigen 1968 if it is assumed that diagonal and adjacent subunits in the same conformation have identical properties. Because of the complexity of the 55- and 35-state schemes, Fig. 1 is often reduced further to the 25-state model described by Fig. 2 (Fersht 1985).

Bottom Line: 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 considered models can serve as working hypotheses for the gating of BK channels.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology and Biophysics, University of Miami School of Medicine, Miami, Florida 33101-6430, USA.

ABSTRACT
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.

Show MeSH