Mutations in voltage-gated sodium channels are linked to several channelopathies leading to a wide variety of diseases including cardiac arrhythmias, epilepsy and myotonia. We previously demonstrated that Na1.5 trafficking-deficient mutant channels could lead to a dominant-negative effect by impairing trafficking of the wild-type (WT) channel. We also reported that voltage-gated sodium channels associate as dimers with coupled gating properties. Here, we hypothesized that the dominant-negative effect of mutant sodium channels could also occur through coupled gating. This was tested using cell surface biotinylation and single-channel recordings to measure the gating probability and coupled gating of the dimers. Co-expression of Na1.5-L325R with WT channels led to a dominant-negative effect as reflected by a 75% reduction in current density. Surprisingly, cell surface biotinylation showed that Na1.5-L325R mutant is capable of trafficking, with 40% of Na1.5-L325R reaching the cell surface when expressed alone. Importantly, even though a dominant-negative effect on the sodium current is observed when WT and Na1.5-L325R are expressed together, the total NaV channel cell surface expression was not significantly altered compared to WT alone. Thus, the trafficking deficiency could not explain the 75% decrease in INa. Interestingly, single-channel recordings showed that Na1.5-L325R exerted a dominant-negative effect on the WT at the gating level. Both coupled gating and gating probability of the WT:L325R dimers were drastically impaired. We conclude that dominant-negative suppression exerted by Na1.5 mutants can also be caused by impairing the WT gating probability, a mechanism resulting from the dimerization and coupled gating of voltage-gated sodium channel α-subunits.

Aims The Brugada syndrome (BrS) is an inherited cardiac disorder predisposing to ventricular arrhythmias. Despite considerable efforts, its genetic basis and cellular mechanisms remain largely unknown. The objective of this study was to identify a new susceptibility gene for BrS through familial investigation. Methods and results Whole-exome sequencing performed in a three-generation pedigree with five affected members allowed the identification of one rare non-synonymous substitution (p.R211H) in RRAD, the gene encoding the RAD GTPase, carried by all affected members of the family. Three additional rare missense variants were found in 3/186 unrelated index cases. We detected higher levels of RRAD transcripts in subepicardium than in subendocardium in human heart, and in the right ventricle outflow tract compared to the other cardiac compartments in mice. The p.R211H variant was then subjected to electrophysiological and structural investigations in human cardiomyocytes derived from induced pluripotent stem cells (iPSC-CMs). Cardiomyocytes derived from induced pluripotent stem cells from two affected family members exhibited reduced action potential upstroke velocity, prolonged action potentials and increased incidence of early afterdepolarizations, with decreased Na+ peak current amplitude and increased Na+ persistent current amplitude, as well as abnormal distribution of actin and less focal adhesions, compared with intra-familial control iPSC-CMs Insertion of p.R211H-RRAD variant in control iPSCs by genome editing confirmed these results. In addition, iPSC-CMs from affected patients exhibited a decreased L-type Ca2+ current amplitude. Conclusion This study identified a potential new BrS-susceptibility gene, RRAD. Cardiomyocytes derived from induced pluripotent stem cells expressing RRAD variant recapitulated single-cell electrophysiological features of BrS, including altered Na+ current, as well as cytoskeleton disturbances.