Intrinsic Voltage Dependence and Ca2+ Regulation of mslo Large Conductance Ca-activated K+ Channels

The kinetic and steady-state properties of macroscopic mslo Ca-activated K+ currents were studied in excised patches from Xenopus oocytes. In response to voltage steps, the timecourse of both activation and deactivation, but for a brief delay in activation, could be approximated by a single exponential function over a wide range of voltages and internal Ca2+ concentrations (Cai). Activation rates increased with voltage and with Cai, and approached saturation at high Cai. Deactivation rates generally decreased with Cai and voltage, and approached saturation at high Cai. Plots of the macroscopic conductance as a function of voltage (G-V) and the time constant of activation and deactivation shifted leftward along the voltage axis with increasing Cai. G-V relations could be approximated by a Boltzmann function with an equivalent gating charge which ranged between 1.1 and 1.8 e as Cai varied between 0.84 and 1,000 microM. Hill analysis indicates that at least three Ca2+ binding sites can contribute to channel activation. Three lines of evidence indicate that there is at least one voltage-dependent unimolecular conformational change associated with mslo gating that is separate from Ca2+ binding. (a) The position of the mslo G-V relation does not vary logarithmically with Cai. (b) The macroscopic rate constant of activation approaches saturation at high Cai but remains voltage dependent. (c) With strong depolarizations mslo currents can be nearly maximally activated without binding Ca2+. These results can be understood in terms of a channel which must undergo a central voltage-dependent rate limiting conformational change in order to move from closed to open, with rapid Ca2+ binding to both open and closed states modulating this central step.