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 KD 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-1, and for defining the intimate relationship between gating and permeation in ClC channels.
Tuning the ion selectivity of tetrameric cation channels by changing the number of ion binding sites
