The voltage-dependent ClC-2 chloride channel has a dual gating mechanism.

Functional and structural studies demonstrate that Cl(-) channels of the ClC family have a dimeric double-barrelled structure, with each monomer contributing an identical pore. Single protopore gating is a fast process dependent on Cl(-) interaction within the selectivity filter and in ClC-0 has a low temperature coefficient over a 10 degrees C range (Q(10)). A slow gating process closes both protopores simultaneously, has a high Q(10), is facilitated by extracellular Zn(2+) and Cd(2+) and is abolished or markedly reduced by mutation of a cysteine conserved in ClC-0, -1 and -2. In order to test the hypothesis that similar slow and fast gates exist in the widely expressed ClC-2 Cl(-) channel we have investigated the effects of these manoeuvres on ClC-2. We find that the time constants of both components of the double-exponential hyperpolarization-dependent activation (and deactivation) processes have a high temperature dependence, with Q(10) values of about 4-5, suggesting important conformational changes of the channel. Mutating C256 (equivalent to C212 in ClC-0) to A, led to a significant fraction of constitutively open channels at all potentials. Activation time constants were not affected but deactivation was slower and significantly less temperature dependent in the C256A mutant. Extracellular Cd(2+), that inhibits wild-type (WT) channels almost fully, inhibited C256A only by 50%. In the WT, the time constants for opening were not affected by Cd(2+) but deactivation at positive potentials was accelerated by Cd(2+). This effect was absent in the C256A mutant. The effect of intracellular Cl(-) on channel activation was unchanged in the C256A mutant. Collectively our results strongly support the hypothesis that ClC-2 possesses a common gate and that part of the current increase induced by hyperpolarization represents an opening of the common gate. In contrast to the gating in ClC-0, the protopore gate and the common gate of ClC-2 do not appear to be independent.