Mechanism of ion permeation in skeletal muscle chloride channels

Voltage-gated Cl^- channels belonging to the ClC family exhibit unique properties of ion permeation and gating. We functionally probed the conduction pathway of a recombinant human skeletal muscle Cl^- channel (hClC- 1) expressed both in Xenopus oocytes and in a mammalian cell line by investigating block by extracellular or intracellular I^- and related anions. Extracellular and intracellular I^- exert blocking actions on hClC-1 currents that are both concentration and voltage dependent. Similar actions were observed for a variety of other halide (Br^-) and polyatomic (SCN^-, NO₃^-, CH₃SO₃^-) anions. In addition, I^- block is accompanied by gating alterations that differ depending on which side of the membrane the blocker is applied. External I^- causes a shift in the voltage-dependent probability that channels exist in three definable kinetic states (fast deactivating, slow deactivating, nondeactivating), while internal I^- slows deactivation. These different effects on gating properties can be used to distinguish two functional ion binding sites within the hClC-1 pore. We determined K(D) values for I^- block in three distinct kinetic states and found that binding of I^- to hClC-1 is modulated by the gating state of the channel. Furthermore, estimates of electrical distance for I^- binding suggest that conformational changes affecting the two ion binding sites occur during gating transitions. These results have implications for understanding mechanisms of ion selectivity in hClC-C, and for defining the intimate relationship between gating and permeation in ClC channels.