Nonequilibrium gating and voltage dependence of the ClC-0 Cl^- channel

The gating of ClC-0, the voltage-dependent Cl^- channel from Torpedo electric organ, is strongly influenced by Cl^- ions in the external solution. Raising external Cl^- over the range 1-600 mM favors the fast-gating open state and disfavors the slow-gating inactivated state. Analysis of purified single ClC-0 channels reconstituted into planar lipid bilayers was used to identify the role of Cl^- ions in the channel's fast voltage-dependent gating process. External, but not internal, Cl^- had a major effect on the channel's opening rate constant. The closing rate was more sensitive to internal Cl^- than to external Cl^-. Both opening and closing rates varied with voltage. A model was derived that postulates (a) that in the channel's closed state, Cl^- is accessible to a site located at the outer end of the conduction pore, where it binds in a voltage-independent fashion, (b) that this closed conformation can open, whether liganded by Cl^- or not, in weakly voltage- dependent fashion, (c) that the Cl^--liganded closed channel undergoes a conformational change to a different closed state, such that concomitant with this change, Cl^- ion moves inward, conferring voltage-dependence to this step, and (d) that this new Cl^--liganded closed state opens with a very high rate. According to this picture, Cl^- movement within the pre-open channel is the major source of voltage dependence, and charge movement intrinsic to the channel protein contributes very little to voltage-dependent gating of ClC- 0. Moreover, since the Cl^- activation site is probably located in the ion conduction pathway, the fast gating of ClC-0 is necessary coupled to ion conduction, a nonequilibrium process.