APPS November 2002 Meeting Abstract 1327

Michael Duffield¹, Grigori Rychkov¹, Allan Bretag²,Michael Roberts¹, 1 Department of Physiology, University of Adelaide, South Australia, 2 Centre for Advanced Biomedical Studies, University of South Australia, South Australia.

The ClC-1 channel is a dimeric, double-pored, chloride channel, predominantly found in skeletal muscle. Mutations in this channel are responsible for the muscle stiffness disorder myotonia. Structurally and mechanistically, members of the ClC channel are quite unlike other families of ion channels, and although a bacterial ClC channel has recently been crystallised, the mechanisms of ClC gating and permeation remain to be elucidated. ClC-1 gating is a complex process, with the channel displaying two different gating processes. This study has used site-directed mutagenesis and electrophysiological techniques to investigate the involvement of the G,H and I α-helical protein domains in slow gating of the ClC-1 channel. These domains lie on the interface of the channel monomers, and contain a number of residues that are mutated in dominant myotonia - a form of myotonia involving alterations in the ClC-1 slow gating process.

Therefore we have produced a number of ClC-1 channels containing point mutations within the G, H or I domains, and investigated the gating of these mutant channels. The majority of these mutations affected slow gating of the ClC-1 channel, without affecting the fast gating process. This occurred through alterations in the opening rate, closing rate, or both, of the slow gate in these mutant channels. These results suggest that mutations in the G, H and I domains affect the energy of the open or closed states of the channel, resulting in alterations in the transition rates between these states. These changes are responsible for the observed alteration in the voltage dependence of the slow gating process that occurs in the mutant channels.