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Centro de Estudios Científicos (CECS), Av. Arturo Prat 514, Casilla 1469, Valdivia, Chile
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°C range (Q10). A slow gating process closes both protopores simultaneously, has a high Q10, is facilitated by extracellular Zn2+ and Cd2+ 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 Q10 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 Cd2+, 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 Cd2+ but deactivation at positive potentials was accelerated by Cd2+. 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.
(Received 18 December 2003;
accepted after revision 9 January 2004;
first published online 14 January 2004)
Corresponding author F. Sepúlveda: Centro de Estudios Científicos (CECS), Av. Arturo Prat 514, Casilla 1469, Valdivia, Chile. Email: fsepulveda{at}cecs.cl
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