Abstract
Brugada syndrome (BrS) is a genetically determined cardiac arrhythmogenic syndrome with increased risk of sudden cardiac death. BrS is mostly caused by mutations in SCN5A gene encoding the primary ɑ-subunit of the cardiac sodium channel Na(V)1.5. We aimed at characterizing the functional alterations caused by the R893C 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 R893, the functional role of this region remains unknown and, in addition, exploring SCN5A 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 R893C almost completely abolished the sodium current, I(Na), though the mutation did not exert dominant-negative effect on wild-type Na(V)1.5 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(V)1.5 in the R893C mutant highlighting a likely mechanism for loss of conduction via formation of disulphide bridges. We showed that R893H channels also failed to produce any detectable I(Na) that confirms the importance of the highly conserved R893 in gating. Our study reveals R893C is a loss-of-function mutation with altered electrophysiological characteristics of Na(V)1.5. Thus, R893C 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(V)1.5 channelopathies.