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 systems. 2. Hyperpolarization induces deactivating inward currents comprising a steady‐state component and two exponentially decaying components, of which the faster is weakly voltage dependent and the slower strongly voltage dependent. 3. Open probability (Po) and kinetics depend on external but not internal Cl‐ concentration. 4. A point mutation, K585E, in human ClC‐1, equivalent to a previously described mutation in the Torpedo electroplaque chloride channel, ClC‐0, alters the I‐V relationship and kinetics, but retains external Cl‐ dependence. 5. When external pH is reduced, the deactivating inward currents of ClC‐1 are diminished without change in time constants while the steady‐state component is enhanced. 6. In contrast, reduced internal pH slows deactivating current kinetics as its most immediately obvious action and the Po curve is shifted in the hyperpolarizing direction. Addition of internal benzoate at low internal pH counteracts both these effects. 7. A current 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 site accessible only from the exterior and, possibly, by modification of this site by external protonation. Intracellular hydroxyl ions strongly affect gating either allosterically or by direct binding and blocking of the pore, an action mimicked by intracellular benzoate.

A distinctive feature of the voltage-dependent chloride channels ClC-0 (the Torpedo electroplaque chloride channel) and ClC-1 (the major skeletal muscle chloride channel) is that chloride acts as a ligand to its own channel, regulating channel opening and so controlling the permeation of its own species. We have now studied the permeation of a number of foreign anions through ClC-1 using voltage-clamp techniques on Xenopus oocytes and Sf9 cells expressing human (hClC-1) or rat (rClC-1) isoforms, respectively. From their effect on channel gating, the anions presented in this paper can be divided into three groups: impermeant or poorly permeant anions that can not replace Cl− as a channel opener and do not block the channel appreciably (glutamate, gluconate, HCO3 −, BrO3 −); impermeant anions that can open the channel and show significant block (methanesulfonate, cyclamate); and permeant anions that replace Cl− at the regulatory binding site but impair Cl− passage through the channel pore (Br−, NO3 −, ClO3 −, I−, ClO4 −, SCN−). The permeability sequence for rClC-1, SCN− ∼ ClO4 − > Cl− > Br− > NO3 − ∼ ClO3 − > I− >> BrO3 − > HCO3 − >> methanesulfonate ∼ cyclamate ∼ glutamate, was different from the sequence determined for blocking potency and ability to shift the P open curve, SCN− ∼ ClO4 − > I− > NO3 − ∼ ClO3 − ∼ methanesulfonate > Br− > cyclamate > BrO3 − > HCO3 − > glutamate, implying that the regulatory binding site that opens the channel is different from the selectivity center and situated closer to the external side. Channel block by foreign anions is voltage dependent and can be entirely accounted for by reduction in single channel conductance. Minimum pore diameter was estimated to be ∼4.5 Å. Anomalous mole-fraction effects found for permeability ratios and conductance in mixtures of Cl− and SCN− or ClO4 − suggest a multi-ion pore. Hydrophobic interactions with the wall of the channel pore may explain discrepancies between the measured permeabilities of some anions and their size.

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Mutations in the ClC-1 muscle chloride channel cause either recessive or dominant myotonia congenita. Using a systematic screening procedure, we have now identified four novel missense mutations in dominant (V286A, F307S) and recessive myotonia (V236L, G285E), and have analysed the effect of these and other recently described mutations (A313T, I556N) on channel properties in the Xenopus oocyte expression system. Mutations V286A, F307S and A313T displayed a 'classical' dominant phenotype: their voltage dependence was shifted towards positive potentials and displayed a dominant-negative effect by significantly imparting a voltage shift on mutant-wild-type heteromeric channels as found in heterozygous patients. In contrast, the recessive mutation V236L also shifted the voltage dependence to positive values, but co-expression with wild-type ClC-1 gave almost wild-type currents. I556N, a mutation found in patients with benign dominant myotonia, drastically shifts the voltage dependence, but only a slight shift is seen when co-expressed with wild-type ClC-1. Thus, the voltage dependence of mutant heteromeric channels is not always intermediate between those of the constituent homomeric channel subunits, a conclusion further supported by mixing different ClC-1 mutants. These complex interactions correlate clinically with various inheritance patterns, ranging from autosomal dominant with various degrees of penetrance to autosomal recessive.