Disease‐causing mutations C277R and C277Y modify gating of human ClC‐1 chloride channels in myotonia congenita

Key points • ClC channels are double‐barrelled channels with two ion conduction pathways per individual channel. • Substituting cysteine 277 by serine constitutively opens the common gate suggesting that this residue plays a major role in joint openings/closings of both protopores of ClC‐1. • We studied here the functional consequences of two novel myotonia‐associated mutations, C277R and C277Y, of human ClC‐1 chloride channels. • C277Y not only modified the common gate, but also protopore gating. • C277Y inverts the voltage dependence and reduces the open probabilities of protopore and common gates and thus decreases the absolute open probabilities of homodimeric hClC‐1 channels to values below 3%. • C277Y reduces single protopore current amplitudes to about two‐thirds of wild‐type values, and inverts the anion permeability sequence. • Our results explain the disease‐causing effects and provide novel insights into the molecular processes underlying normal and pathologically altered function of muscle chloride channels. Myotonia congenita is a genetic condition that is caused by mutations in the muscle chloride channel gene CLCN1 and characterized by delayed muscle relaxation and muscle stiffness. We here investigate the functional consequences of two novel disease‐causing missense mutations, C277R and C277Y, using heterologous expression in HEK293T cells and patch clamp recording. Both mutations reduce macroscopic anion currents in transfected cells. Since hClC‐1 is a double‐barrelled anion channel, this reduction in current amplitude might be caused by altered gating of individual protopores or of joint openings and closing of both protopores. We used non‐stationary noise analysis and single channel recordings to separate the mutants’ effects on individual and common gating processes. We found that C277Y inverts the voltage dependence and reduces the open probabilities of protopore and common gates resulting in decreases of absolute open probabilities of homodimeric channels to values below 3%. In heterodimeric channels, C277R and C277Y also reduce open probabilities and shift the common gate activation curve towards positive potentials. Moreover, C277Y modifies pore properties of hClC‐1. It reduces single protopore current amplitudes to about two‐thirds of wild‐type values, and inverts the anion permeability sequence to I−= NO3− > Br− > Cl−. Our findings predict a dramatic reduction of the muscle fibre resting chloride conductance and thus full