ClC‐1 mutations in myotonia congenita patients: insights into molecular gating mechanisms and genotype–phenotype correlation

Abstract

Key pointsr Loss-of-function mutations of the skeletal muscle ClC-1 channel cause myotonia congenita with variable phenotypes.r Using patch clamp we show that F484L, located in the conducting pore, probably induces mild dominant myotonia by right-shifting the slow gating of ClC-1 channel, without exerting a dominant-negative effect on the wild-type (WT) subunit.r Molecular dynamics simulations suggest that F484L affects the slow gate by increasing the frequency and the stability of H-bond formation between E232 in helix F and Y578 in helix R.r Three other myotonic ClC-1 mutations are shown to produce distinct effects on channel function: L198P shifts the slow gate to positive potentials, V640G reduces channel activity, while L628P displays a WT-like behaviour (electrophysiology data only).r Our results provide novel insight into the molecular mechanisms underlying normal and altered ClC-1 function.Abstract Myotonia congenita is an inherited disease caused by loss-of-function mutations of the skeletal muscle ClC-1 chloride channel, characterized by impaired muscle relaxation after contraction and stiffness. In the present study, we provided an in-depth characterization of F484L, a mutation previously identified in dominant myotonia, in order to define the genotype-phenotype correlation, and to elucidate the contribution of this pore residue to the mechanisms of ClC-1 gating. Patch-clamp recordings showed that F484L reduced chloride currents at every tested potential and dramatically right-shifted the voltage dependence of slow gating, thus contributing to the mild clinical phenotype of affected heterozygote carriers. Unlike dominant mutations located at the dimer interface, no dominant-negative effect was observed when F484L mutant subunits were co-expressed with wild type. Molecular dynamics simulations further revealed that F484L affected the slow gate by increasing the frequency and stability of the H-bond formation between the pore residue E232 and the R helix residue Y578. In addition, using patch-clamp electrophysiology, we characterized three other myotonic ClC-1 mutations. We proved that the dominant L198P mutation in the channel pore also right-shifted the voltage dependence of slow gating, recapitulating mild myotonia. The recessive V640G mutant drastically reduced channel function, which probably accounts for myotonia. In contrast, the recessive L628P mutant produced currents very similar to wild type, suggesting that the occurrence of the compound truncating mutation (Q812X) or other muscle-specific mechanisms accounted for the severe symptoms observed in this family. Our results provide novel insight into the molecular mechanisms underlying normal and altered ClC-1 function.

The analysis of myotonia congenita mutations discloses functional clusters of amino acids within the CBS2 domain and the C-terminal peptide of the ClC-1 channel

“…In hClC‐1, the CBS2 domain encompasses residues 820 to 871 (Estévez et al., ; Hebeisen et al., ) and has been predicted to play a role in channel gating. Indeed, structural studies on ClC‐1 suggested that the long intracellular C‐terminal portion is folded in such a manner that the CBS2 domain may directly interact with the α‐helix R containing Tyr578 residue, which participates directly in Cl − coordination during transport (Feng et al., ; Imbrici et al., ). Moreover, several mutations occurring in CBS2 have been shown to cause changes in channel voltage dependence, and a mutant with a particularly strong effect, p.His835Arg, displayed a dominant‐negative effect on WT in coexpression studies, suggesting an alteration of the common gate (Estévez et al., ).…”

“…Open probability for common gating ( P o common) was obtained by using a similar protocol to that for the overall P o (200 ms conditioning pulse), except that an extra 400 μs activation pulse to +180 mV was added before stepping to −105 mV (Imbrici et al., , ). This very positive step fully activates the fast gates of the channel and the tail currents at −105 mV then reflect only slow gating.…”

“…Open probability for common gating (P o common) was obtained by using a similar protocol to that for the overall P o (200 ms conditioning pulse), except that an extra 400 s activation pulse to +180 mV was added before stepping to −105 mV (Imbrici et al, 2015b(Imbrici et al, , 2016b.…”

“…In hClC-1, the CBS2 domain encompasses residues 820 to 871 (Estévez et al, 2004;Hebeisen et al, 2004) and has been predicted to play a role in channel gating. Indeed, structural studies on ClC-1 suggested that the long intracellular C-terminal portion is folded in such a manner that the CBS2 domain may directly interact with the -helix R containing Tyr578 residue, which participates directly in Cl − coordination during transport (Feng et al, 2010;Imbrici et al, 2015b poor expression due to a defect in protein translocation to the membrane. Interestingly, p.Gly859Asp is also associated with a lack of current, suggesting presumably a similar disruption of the CBS domain structure (Bennetts et al, 2005).…”

A case of Becker myotonia with pseudodominant inheritance: сurrent approaches to the differential diagnosis of Thomsen’s and Becker's myotonia congenita

“…Все клинические проявления находились в зоне перекрытия фенотипов МТ и МБ, что не способствовало уточнению нозоло-гии. Опираться только на клинические признаки при дифференциальной диагностике МТ и МБ нельзя, так как в одной семье у больных с одинаковыми мутациями выраженность симптомов варьирует в широких преде-лах, а в последних обзорах все чаще авторы не находят статистически значимых клинических отличий между МТ и МБ [7,[19][20][21][22].…”