Characterization of the R893C NaV1.5 mutation in Brugada syndrome

Brugada syndrome (BrS) is a genetically determined cardiac arrhythmogenic syndrome with increased risk of sudden cardiac death. BrS is mostly caused by mutations in <i>SCN5A</i> gene encoding the primary ɑ-subunit of the cardiac sodium channel Na_V1.5. We aimed at characterizing the functional alterations caused by the <i>R893C</i> mutation, identified in a proband diagnosed with BrS, and establishing whether the mutation is associated with BrS. Although several mutations have been reported in the close vicinity of <i>R893</i>, the functional role of this region remains unknown and, in addition, exploring <i>SCN5A</i> mutations in patients with inherited arrhythmogenic syndromes is critical for understanding the pathogenesis of arrhythmias. The mutations were introduced by site-directed mutagenesis. The variants were transiently expressed in CHO cells and potassium currents were measured using the whole-cell patch clamp technique. Patch clamp recordings have demonstrated that <i>R893C</i> almost completely abolished the sodium current, I_Na, though the mutation did not exert dominant-negative effect on wild-type Na_V1.5 channels. We also observed significant decrease in channel activation and a depolarized shift of steady-state inactivation curve, however, the kinetics of inactivation and recovery from fast inactivation were not changed by the mutation. Moreover, the reducing agent Dithiotreitol partially restored the normal function of Na_V1.5 in the <i>R893C</i> mutant highlighting a likely mechanism for loss of conduction via formation of disulphide bridges. We showed that <i>R893H</i> channels also failed to produce any detectable I_Na that confirms the importance of the highly conserved <i>R893</i> in gating. Our study reveals <i>R893C</i> is a loss-of-function mutation with altered electrophysiological characteristics of Na_V1.5. Thus, <i>R893C</i> may contribute to the BrS phenotype of the proband. Our findings may facilitate the understanding of the mechanisms of arrhythmogenesis in BrS, as it helps to identify mutational hotspots in BrS. Moreover, our work may improve novel gene therapy and new therapeutic drug design targeting Na_V1.5 channelopathies.