RESUMEN
Myotonia is a condition characterized by impaired relaxation of muscle following sudden forceful contraction. We systematically screened all 23 exons of the CLCN1 gene in 88 unrelated patients with myotonia and identified mutations in 14 patients. Six novel mutations were discovered: five were missense (S132C, L283F, T310M, F428S and T550M) found in heterozygous patients, and one was a nonsense mutation (E193X) in a homozygous patient. While five patients had a clinical diagnosis of myotonia congenita, the patient with the F428S mutation exhibited symptoms characteristic of paramyotonia congenita--a condition usually thought to be caused by mutations in the sodium channel gene SCN4A. Nevertheless, no mutations in SCN4A were identified in this patient. The functional consequences of the novel CLCN1 sequence variants were explored by recording chloride currents from human embryonic kidney cells transiently expressing homo- or heterodimeric mutant channels. The five tested mutations caused distinct functional alterations of the homodimeric human muscle chloride ion channel hClC-1. S132C and T550M conferred novel hyperpolarization-induced gating steps, L283F and T310M caused a shift of the activation curve to more positive potentials and F428S reduced the expression level of hClC-1 channels. All showed a dominant-negative effect. For S132C, L283F, T310M and T550M, heterodimeric channels consisting of one wild-type (WT) and one mutant subunit exhibited a shifted activation curve at low intracellular [Cl(-)]. WT-F428S channels displayed properties similar to WT hClC-1, but expressed at significantly lower levels. The novel mutations exhibit a broad variety of functional defects that, by distinct mechanisms, cause a significant reduction of the resting chloride conductance in muscle of heterozygous patients. Our results provide novel insights into functional alterations and clinical symptoms caused by mutations in CLCN1.