The durations of adjacent open and shut intervals, obtained with the patch-clamp technique from fast Cl- channels in tissue-cultured rat skeletal muscle, were analysed to distinguish among eight previously considered gating mechanisms for the channel which differed in the connections among the states. Open intervals were separated into groups based on the duration of the shut intervals which occurred before or after each open interval. Fitting these conditional open distributions with sums of exponentials indicated that they were described by two exponential components. The time constants of the two components in the conditional open distributions were independent of the adjacent shut interval durations. The observation of invariant time constants is consistent with gating mechanisms in which the rate constants for transitions among the states remain constant with time (discrete Markow process). In contrast to the invariant time constants, the areas of the two components in the conditional open distributions were dependent on the adjacent shut interval durations. The area of the fast open component increased, and the area of the slow open component decreased, as the duration of adjacent shut intervals increased. Thus, it is changes in areas, rather than time constants, which give rise to the observed inverse relationship between the durations of adjacent open and shut intervals. The findings in summary statements 2-4 indicate that at least two open states are connected by independent pathways to different shut states; the open state associated with the fast open component is connected to a shut state (or compound shut state) of longer effective lifetime, and the open state associated with the slow open component is connected to a shut state (or compound shut state) of briefer effective lifetime. Seven of the eight previously considered gating mechanisms were rejected because they did not account for the observed relationships between the durations of adjacent open and shut intervals, when analysed in terms of either conditional open distributions or conditional mean open interval durations. The seven rejected gating mechanisms also did not account for the observed correlations between interval durations, when analysed in terms of correlation coefficients. Adjacent interval and correlation analysis thus provided a means to distinguish among the gating mechanisms. A free energy state diagram derived, assuming absolute rate theory, from the rate constants for the one consistent kinetic scheme indicated that the free energy required to overcome the effective transition barriers between states was 11-15 kcal/mol, whereas the free energy difference between connected equilibrium states was equal to only about 0.5-1.5 kcal/mol. Thus, if transitions between states involve the breaking of weak bonds (hydrogen bonds, van der Waals bonds or salt bridges), then about the same equivalent number of bonds are reformed immediately after each transition.
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