Gating Kinetics of Single Large - Conductance Ca 2 1 - activated K 1 Channels in High Ca 2 1 Suggest a Two - Tiered Allosteric Gating Mechanism ✪

The Ca 2 1 -dependent gating mechanism of large-conductance calcium-activated K 1 (BK) channels from cultured rat skeletal muscle was examined from low (4 m M) to high (1,024 m M) intracellular concentrations of calcium (Ca 2 1 i ) using single-channel recording. Open probability ( P o ) increased with increasing Ca 2 1 i ( K 0.5 11.2 6 0.3 m M at 1 30 mV, Hill coefficient of 3.5 6 0.3), reaching a maximum of z 0.97 for Ca 2 1 i z 100 m M. Increasing Ca 2 1 i further to 1,024 m 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 2 1 i ( . 100 m M), compared with three to four open and five to seven closed states at lower Ca 2 1 i . The ability of kinetic schemes to account for the single-channel kinetics was examined with simultaneous maximum likelihood fitting of twodimensional (2-D) dwell-time distributions obtained from low to high Ca 2 1 i . Kinetic schemes drawn from the 10state Monod-Wyman-Changeux model could not describe the dwell-time distributions from low to high Ca 2 1 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 Ca 2 1 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 Ca 2 1 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 Ca 2 1 i . A model that assumed that the apparent Ca 2 1 -binding steps can reach a maximum rate at high Ca 2 1 i could also approximate the gating from low to high Ca 2 1 i . The considered models can serve as working hypotheses for the gating of BK channels. key words: BK channel • K Ca channel • Monod-Wyman-Changeux • Eigen • Markov i n t r o d u c t i o n Large-conductance Ca 2 1 -activated K 1 (BK) 1 channels, which are activated by micromolar concentrations of intracellular Ca 2 1 (Ca 2 1 i ) and by depolarization (Barrett et al., 1982) are present in a wide variety of tissues (reviewed by Rudy, 1988; Marty, 1989; McManus, 1991; Latorre, 1994; Conly, 1996). Activated BK channels reduce membrane excitability by allowing K 1 efflux through their opened pores, which drives the membrane potential more negative. Hence, BK channels form a link in a negative feed-back loop that decreases excitability in response to both increased Ca 2 1 i and depolarization. Information about the gating mechanism of BK channels has accumulated from kinetic studies on native and heterologously expressed channels. BK channels are homotetramers, formed from four alpha subunits (Shen et al., 1994), with one or more Ca 2 1 -binding sites per subunit (Schreiber and Salkoff, 1997). BK channels typically display a steep relation between Ca 2 1 i and open probability, P o , with Hill coefficients usually in the range of 2–4 (Barrett et al. 1982; Wong et al., 1982; Golowasch et al., 1986; Oberhauser et al., 1988; Reinhart et al., 1989; McManus and Magleby, 1991; Art et al., 1995; DiChiara and Reinhart, 1995; Cui et al., 1997). Hill coefficients . 1 suggest a cooperative action of Ca 2 1 on P o , and are often associated with allosteric modulation of activity (Colquhoun 1973; Fersht 1985). Models drawn from the Monod-Wyman-Changeux (MWC) model (Monod et al., 1965) describing the transitions of tetrameric allosteric proteins have often been used to describe the Ca 2 1 -dependent gating of BK channels. For example, McManus and Magleby (1991) found that a model with three open and five closed states drawn from the MWC model could account for many of the basic features of the Ca 2 1 dependence of the single-channel gating kinetics from low to intermediate levels of activity, and Cox et al. (1997b) found that the full 10-state MWC model (and also an extended 12state model) could account for macroscopic conductance–voltage relations over a wide range of Ca 2 1 i . Address correspondence to Dr. Karl L. Magleby, Dept. of Physiology and Biophysics (R-430), University of Miami School of Medicine, Miami, FL 33101-6430. Fax: 305-243-6898; E-mail: kmagleby@