G.P.100 Biophysical characterization of hClC-1 chloride channel mutations associated with myotonia congenita

Abstract

Abstract Myotonia congenita (MC) is an inherited skeletal muscle disease characterized by muscle stiffness during sudden forceful movement, due to electrical instability of the sarcolemma. MC is due to loss-of-function mutations in CLCN1 gene encoding the ClC-1 chloride channel. Three CLCN1 mutations (G190S, A531V and F167L) individuated in MC patients were introduced in hClC-1 using standard site-directed mutagenesis. Recombinant wild-type and mutant hClC-1 channels were expressed in HEK cells to study chloride currents using the whole-cell patch-clamp technique. A531V induced a large decrease of chloride current density but the voltage-dependence of open probability (Po) and deactivation kinetics were similar to WT. Instead, G190S determined a dramatic positive shift by ∼180 mV of the Po voltage-dependence in physiological chloride gradient. Although the biophysical mechanism were different, the dramatic reduction of A531V and G190S chloride currents fully explain their role in determining MC. Conversely, whatever the chloride ion concentration, F167L induced no significant change in chloride current density, kinetics, and voltage dependence with respect to WT. In order to understand how F167L channels may cause MC, we characterized the compound mutations found in patients carrying F167L, namely G355R, G284R, Y686X, P558S and R105C. The G284R, G355R and Y686X mutations generated no current in HEK cells, suggesting that they constitute the main contributor to MC phenotype in heterozygous patients. Whereas R105C produced chloride currents very similar to WT, P558S profoundly affected chloride current kinetics and voltage dependence. Nevertheless, both mutations reduced chloride current density in physiological chloride gradient, confirming their guilty role in MC. Ongoing co-expression experiments are performed to evaluate the possible interaction between compound mutations. Supported by Telethon-Italy (GGP10101) and Association Francaise contre les Myopathies (15020).