Insights into CLC-0's Slow-Gating from Intracellular Proton Inhibition

The opening of the Torpedo CLC-0 chloride (Cl^-) channel is known to be regulated by two gating mechanisms: fast gating and slow (common) gating. The structural basis underlying the fast-gating mechanism is better understood than that of the slow-gating mechanism, which is still largely a mystery. Our previous study on the intracellular proton (H⁺_i)-induced inhibition of the CLC-0 anionic current led to the conclusion that the inhibition results from the slow-gate closure (also called inactivation). The conclusion was made based on substantial evidence such as a large temperature dependence of the H⁺_i inhibition similar to that of the channel inactivation, a resistance to the H⁺_i inhibition in the inactivation-suppressed C212S mutant, and a similar voltage dependence between the current recovery from the H⁺_i inhibition and the recovery from the channel inactivation. In this work, we further examine the mechanism of the H⁺_i inhibition of wild-type CLC-0 and several mutants. We observe that an anion efflux through the pore of CLC-0 accelerates the recovery from the H⁺_i-induced inhibition, a process corresponding to the slow-gate opening. Furthermore, various inactivation-suppressed mutants exhibit different current recovery kinetics, suggesting the existence of multiple inactivated states (namely, slow-gate closed states). We speculate that protonation of the pore of CLC-0 increases the binding affinity of permeant anions in the pore, thereby generating a pore blockage of ion flow as the first step of inactivation. Subsequent complex protein conformational changes further transition the CLC-0 channel to deeper inactivated states.