Intrinsic Voltage Dependence and Ca ²⁺ Regulation of mslo Large Conductance Ca-activated K ⁺ Channels

Abstract

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 Ca ²⁺ concentrations ([Ca] _i). Activation rates increased with voltage and with [Ca] _i, and approached saturation at high [Ca] _i. Deactivation rates generally decreased with [Ca] _i and voltage, and approached saturation at high [Ca] _i. 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 [Ca] _i. 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 [Ca] _i varied between 0.84 and 1,000 μM. Hill analysis indicates that at least three Ca ²⁺ 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 Ca ²⁺ binding. ( a) The position of the mslo G-V relation does not vary logarithmically with [Ca] _i. ( b) The macroscopic rate constant of activation approaches saturation at high [Ca] _i but remains voltage dependent. ( c) With strong depolarizations mslo currents can be nearly maximally activated without binding Ca ²⁺. 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 Ca ²⁺ binding to both open and closed states modulating this central step.