The voltage dependence and stability of the gating kinetics of the fast Cl- channel in excised patches of membrane from cultured rat skeletal muscle were studied with the patch clamp technique. Up to 106 open and shut intervals were analysed from each of five different patches containing a single fast Cl- channel. To test for kinetic stability, plots of the mean durations of consecutive groups of 5-500 open and shut intervals were examined at each voltage. After excluding infrequent entries into both an apparent subconductance state and long-lived (inactive) shut state, there were no abrupt and sustained changes in the moving means, indicating the absence of obvious shifts to other kinetic modes. The moving means did, however, fluctuate about the overall mean. A comparison of experimental and simulated data indicated that most, but not all, of the fluctuation in the moving means was due to the stochastic variation inherent in the gating process. The fluctuation not accounted for by stochastic variation was mainly expressed as a slow, low-amplitude, component of drift about the mean. This slow component was unlikely to have arisen from measurement errors. To examine whether the slow drift reflected detectable changes in kinetic modes, the data were divided into consecutive groups of 50000 intervals. The exponential components describing the distributions were remarkably similar among the different groups, with stochastic variation accounting for most of the observed differences. This finding implies a single kinetic mode throughout the experiment. Thus, any changes in channel activity associated with the slow drift would have little effect on the analysis of gating kinetics presented here. Depolarization increased channel open probability, P(open), for all five channels. This increase had a voltage sensitivity of 17 ± 4 mV per e-fold change (effective gating charge of 1.6 ± 0.32 electronic charges at 23°C). P(open) was 0.5 at -31 ± 4 mV. The depolarization induced increase in P(open) typically arose from a decrease in the mean shut time (19 ± 4 mV per e-fold change; effective gating charge of 1.3 ± 0.3 at 23°C) and an increase in the mean open time (109 ± 61 mV per e-fold change; effective gating charge of -0.24 ± 0.13). Neither plots of P(open) versus voltage nor plots of the mean open and mean shut time versus voltage were completely described by a single Boltzmann distribution, suggesting multiple voltage-sensitive steps in channel gating. The number of exponential components in the open and shut dwell-time distributions (typically two open and six shut) were independent of membrane potential (examined range: -30 to -100 mV), suggesting that the voltage dependence of P(open) does not result from a voltage-dependent change in the effective number of kinetic states. The typical effect of depolarization on the open dwell-time distribution was to increase the time constants of both exponential components and shift area from the fast to the slow component. The typical effect of depolarization on the shut dwell-time distribution was to decrease the time constants of all six components and shift net area from the four slower to the fastest component. For one of the five channels the observed mean open time decreased, rather than increased, with depolarization. This reversed voltage sensitivity of the observed mean open time may actually reflect a reversed voltage sensitivity of a rate away from a shut state. In addition, there were some differences in some of the other kinetic parameters among the channels. These differences suggest some kinetic heterogeneity, but the heterogeneity was small relative to the overall similarity in gating kinetics of the five channels. The typical results suggest a kinetic scheme for the normal activity of the fast Cl- channel with two open and six shut states. All kinetic states are entered at all the examined voltages, with the frequency of entry and the lifetimes of the various states shifting progressively with depolarization to generate longer mean open intervals and briefer mean shut intervals.
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