Abstract
Large-conductance Ca(2+)-activated K(+) channels can be activated by membrane voltage in the absence of Ca(2+) binding, indicating that these channels contain an intrinsic voltage sensor. The properties of this voltage sensor and its relationship to channel activation were examined by studying gating charge movement from mSlo Ca(2+)-activated K(+) channels in the virtual absence of Ca(2+) (<1 nM). Charge movement was measured in response to voltage steps or sinusoidal voltage commands. The charge-voltage relationship (Q-V) is shallower and shifted to more negative voltages than the voltage-dependent open probability (G-V). Both ON and OFF gating currents evoked by brief (0.5-ms) voltage pulses appear to decay rapidly (tau(ON) = 60 microseconds at +200 mV, tau(OFF) = 16 microseconds at -80 mV). However, Q(OFF) increases slowly with pulse duration, indicating that a large fraction of ON charge develops with a time course comparable to that of I(K) activation. The slow onset of this gating charge prevents its detection as a component of I(gON), although it represents approximately 40% of the total charge moved at +140 mV. The decay of I(gOFF) is slowed after depolarizations that open mSlo channels. Yet, the majority of open channel charge relaxation is too rapid to be limited by channel closing. These results can be understood in terms of the allosteric voltage-gating scheme developed in the preceding paper (Horrigan, F.T., J. Cui, and R.W. Aldrich. 1999. J. Gen. Physiol. 114:277-304). The model contains five open (O) and five closed (C) states arranged in parallel, and the kinetic and steady-state properties of mSlo gating currents exhibit multiple components associated with C-C, O-O, and C-O transitions.
Highlights
K e y wor d s : calcium potassium channel BK channel ion channel gating gating current introduction
Reduction of the sinwave amplitude from 30 to 3 mV had no detectable effect on the charge movement (Cg)–V relationship (Fig. 1 D). This result suggests that the Cg–V was not distorted by the size of the sinusoidal command and is consistent with the weak voltage dependence of mouse homologue of the Slo gene (mSlo) channel gating
Comparison of the Qg–V and GK–V relationships (Fig. 1 B) suggests that charge movement can occur at voltages where most channels are closed
Summary
Experiments were performed with the mbr clone of the mouse homologue of the Slo gene (mSlo), kindly provided by Dr Larry. “0 Ca2ϩ” solutions contained 2 mM EGTA, reducing free Ca2ϩ to an estimated 0.8 nM in the presence of ف10 M contaminant Ca2ϩ (Cox et al, 1997). Data were acquired with an Axopatch 200B amplifier (Axon Instruments, Inc.) in patch mode at a relatively low gain (1–2 mV/pA) to avoid saturation of capacitive transients in response to voltage steps that often exceeded 300 mV. Both the voltage command and current output were filtered at 20 kHz with 8-pole bessel filters (Frequency Devices, Inc.) to limit the speed of fast capacitive transients so that they could be accurately sampled and subtracted. Voltage commands and simulated currents were convolved with the impulse response of a 20 kHz 8-pole bessel filter to reproduce the experimental condition (see Horrigan et al, 1999)
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