Mechanism of inverted activation of ClC-1 channels caused by a novel myotonia congenita mutation

The voltage-gated chloride channel ClC-1 is the major contributor of membra ne conductance in skeletal muscle and has been associated with the inherite d muscular disorder myotonia congenita. Here, we report a novel mutation id entified in a recessive myotonia congenita family, This mutation, Gly-499 t o Arg (G499R) is located in the putative transmembrane domain 10 of the ClC -1 protein. In contrast to normal ClC-1 channels that deactivate upon hyper polarization, functional expression of G499R ClC-1 yielded a hyperpolarizat ion-activated chloride current when measured in the presence of a high (134 mM) intracellular chloride concentration. Current was abolished when measu red with a physiological chloride transmembrane gradient. Electrophysiologi cal analysis of other Gly-499 mutants (G499K, G499Q, and G499E) suggests th at the positive charge introduced by the G499R mutation may be responsible for this unique gating behavior. To further explore the function of domain 10, we mutated two charged residues near Gly-499 of ClC-1. Functional analy ses of R496Q, R496Q/G499R, R496K, and E500Q mutant channels suggest that th e charged residues in domain 10 are important for normal channel function. Study of these mutants may shed further light on the structure and voltage- gating of this channel.