Intrinsic Voltage Dependence and Ca2+ 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 Ca2+ 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 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 [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 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.

Resting Potential and Submembrane Calcium Concentration of Inner Hair Cells in the Isolated Mouse Cochlea Are Set by KCNQ-Type Potassium Channels

“…6A,B). Similar to previous reports (Cui et al, 1997), the increase in [Ca 2ϩ ] i resulted in a decreased time constant for channel activation (Fig. 6A).…”

“…these channels, however, was activated when V R was shifted to depolarized potentials as a result of block of the KCNQ4 channels. Activation of BK channels at potentials as negative as Ϫ65 mV is thought to require [Ca 2ϩ ] i in the upper micromolar range (Adelman et al, 1992;Cui et al, 1997), suggesting that the linopirdine-induced depolarization led to a voltage-dependent increase in [Ca 2ϩ ] i , most likely arising from the activation of voltage-gated Ca 2ϩ channels (Ca V ). Consistent with this idea, a hallmark of IHC Ca 2ϩ channels (␣1D L-type Ca 2ϩ channels, Ca V 1.3) is their unusually negative activation range (Platzer et al, 2000;Xu and Lipscombe, 2001).…”

“…This current is carried by the largeconductance Ca 2ϩ -and voltage-activated BK channels, as indicated by its sensitivity to the BK blockers iberiotoxin and charybdotoxin and its submillisecond activation and deactivation kinetics (Kros and Crawford, 1990;Kros et al, 1998). In this study, we used BK channels as sensors to estimate [Ca 2ϩ (Cui et al, 1997). Although all BK channels are transcribed from a single gene locus, Slo, their Ca 2ϩ sensitivity differs between cell types because of extensive alternative splicing (Tseng-Crank et al, 1994), association with ␤-subunits (McManus et al, 1995; Brenner et al, 2000), and protein phosphorylation (Levitan, 1994;Hall and Armstrong, 2000).…”

Large conductance Ca2+-activated K+ (BK) channel: Activation by Ca2+ and voltage

“…4C. This type of plot was extensively used in the characterization of BK channels and gave the first hint of the existence of a low affinity Ca 2+ site (Wei et al, 1994;Cui et al, 1997). Fig.…”

“…It would be interesting to test for the possibility that the low Ca 2+ affinity of these putative Ca 2+ and Mg 2+ sites is determined by the close proximity of the histidine residues. Despite the fact that it contains an S4 domain, the molecular nature of the BK channel voltage-dependence remained unclear until it was unequivocally demonstrated that depolarizing voltages are able to activate the BK channel in the absence of Ca 2+ (Pallota, 1985;Meera et al, 1996;Cui et al, 1997;Cox et al, 1997;Stefani et al, 1997;Rothberg and Magleby, 2000;Nimigean and Magleby, 2000;Taludker and Aldrich, 2000). One of the first observations in support of the presence of an integral voltage sensor in the BK channel-forming protein is depicted in Figure 4B.…”