CONCENTRATION AND PH-DEPENDENCE OF SKELETAL-MUSCLE CHLORIDE CHANNEL CLC-1

1. The influence of Cl- concentration and pH on gating of the skeletal muscle Cl- channel, ClC-1, has been assessed using the voltage-clamp technique and the Sf-9 insect cell and Xenopus oocyte expression syste ms. 2. Hyperpolarization induces deactivating inward currents comprisi ng a steady-state component and two exponentially decaying components, of which the faster is weakly voltage dependent and the slower strong ly voltage dependent. 3. Open probability (P-o) and kinetics depend on external but not internal Cl- concentration. 4. A point mutation, K58 5E, in human ClC-1, equivalent to a previously described mutation in t he Torpedo electroplaque chloride channel, ClC-0, alters the I-V relat ionship and kinetics, but retains external Cl- dependence. 5. When ext ernal pH is reduced, the deactivating inward currents of ClC-1 are dim inished without change in time constants while the steady-state compon ent is enhanced. 6. In contrast, reduced internal pH slows deactivatin g current kinetics as its most immediately obvious action and the P-o curve is shifted in the hyperpolarizing direction. Addition of interna l benzoate at low internal pH counteracts both these effects. 7. A cur rent activated by hyperpolarization can be revealed at an external pH of 5.5 in ClC-1, which in some ways resembles currents due to the slow gates of ClC-0. 8. Gating appears to be controlled by a Cl--binding s ite accessible only from the exterior and, possibly, by modification o f this site by external protonation. Intracellular hydroxyl ions stron gly affect gating either allosterically or by direct binding and block ing of the pore, an action mimicked by intracellular benzoate.