Fast and slow gating relaxations in the muscle chloride channel CLC-1

Gating of the muscle chloride channel CLC-1 involves at least two processes evidenced by double-exponential current relaxations when stepping the volt age to negative values. However, there is little information about the gati ng of CLC-1 at positive voltages. Here, we analyzed macroscopic gating of C LC-1 over a large voltage range (from - 160 to +200 mV). Activation was fas t at positive voltages but could be easily followed using envelope protocol s that employed a tail pulse to -140 mV after stepping the voltage to a cer tain test potential for increasing durations. Activation was biexponential, demonstrating the presence of two gating processes. Both time constants be came exponentially faster at positive voltages. A similar voltage dependenc e was also seen for the fast gate time constant of CLC-0. The voltage depen dence of the time constant of the fast process of CLC-1, tau(f), was steepe r than that of the slow one, tau(s) (apparent activation valences were z(f) similar to -0.79 and z(s) similar to -0.42) such that at +200 mV the two p rocesses became kinetically distinct by almost two orders of magnitude (tau (f) similar to 16 mu s, tau(s) similar to 1 ms). This voltage dependence is inconsistent with a previously published gating model for CLC-1 (Fahlke, C ., A. Rosenbohm, N. Mitrovic, A.L. George, and R. Rudel. 1996. Biophys. J. 71:695-706). The kinetic difference at 200 mV allowed us to separate the st eady state open probabilities of the two processes assuming that they refle ct two parallel (not necessarily independent) gates that have to be open si multaneously to allow ion conduction. Both open probabilities could be desc ribed by Boltzmann functions with gating valences around one and with nonze ro "offsets" at negative voltages, indicating that the true "gates" never c lose completely. For comparison with single channel data and to correlate t he two gating processes with the two gates of CLC-0, we characterized their voltage, pH(int), and [Cl](ext) dependence, and the dominant myotonia indu cing mutation, I290M. Assuming a double-barreled structure of CLC-1, our re sults are consistent with the identification of the fast and slow gating pr ocesses with die single-pore and the common-pore gate, respectively.