The heartbeat is initiated by voltage-gated sodium channel NaV1.5, which opens rapidly and triggers the cardiac action potential; however, the structural basis for pore opening remains unknown. Here, we blocked fast inactivation with a mutation and captured the elusive open-state structure. The fast inactivation gate moves away from its receptor, allowing asymmetric opening of pore-lining S6 segments, which bend and rotate at their intracellular ends to dilate the activation gate to ∼10 Å diameter. Molecular dynamics analyses predict physiological rates of Na+ conductance. The open-state pore blocker propafenone binds in a high-affinity pose, and drug-access pathways are revealed through the open activation gate and fenestrations. Comparison with mutagenesis results provides a structural map of arrhythmia mutations that target the activation and fast inactivation gates. These results give atomic-level insights into molecular events that underlie generation of the action potential, open-state drug block, and fast inactivation of cardiac sodium channels, which initiate the heartbeat.

Objective: Voltage-gated sodium channel Nav1.5 encoded by the SCN5A gene plays crucial roles in cardiac electrophysiology. Previous genetic studies have shown that mutations in SCN5A are associated with multiple inherited cardiac arrhythmias. Here, we investigated the molecular defect in a Chinese boy with clinical manifestations of arrhythmias. Methods: Gene variations were screened using whole-exome sequencing and validated by direct Sanger sequencing. A minigene assay and reverse transcription PCR (RT-PCR) were performed to confirm the effects of splice variants in vitro. Western blot analysis was carried out to determine whether the c.2262+3A>T variant produced a truncated protein. Results: By genetic analysis, we identified a novel splice variant c.2262+3A>T in SCN5A gene in a Chinese boy with incessant ventricular tachycardias (VT). This variant was predicted to activate a new cryptic splice donor site and was identified by in silico analysis. The variant retained 79 bp at the 5\' end of intron 14 in the mature mRNA. Furthermore, the mutant transcript that created a premature stop codon at 818 amino acids [p.(R818*)] could be produced as a truncated protein. Conclusion: We verified the pathogenic effect of splicing variant c.2262+3A>T, which disturbed the normal mRNA splicing and caused a truncated protein, suggesting that splice variants play an important role in the molecular basis of early onset incessant ventricular tachycardias, and careful molecular profiling of these patients will be essential for future effective personalized treatment options.

BACKGROUND: We have identified the cardiomyopathy-susceptibility gene vinculin (VCL) mutation M94I may account for a sudden unexplained nocturnal death syndrome (SUNDS) case. We addressed whether VCL common variant D841H is associated with SUNDS.
RESULTS: In 8 of 120 SUNDS cases, we detected an East Asian common VCL variant p.Asp841His (D841H). Comparing the H841 allele frequency of the general population in the local database (15 of 1818) with SUNDS victims (10 of 240) gives an odds ratio for SUNDS of 5.226 (95% CI, 2.321, 11.769). The VCL-D841H variant was engineered and either coexpressed with cardiac sodium channel (SCN5A) in HEK293 cells or overexpressed in human induced pluripotent stem-cell-derived cardiomyocytes to examine its effects on sodium channel function using the whole-cell patch-clamp method. In HEK293 cells, under physiological pH conditions (pH 7.4), D841H caused a 29% decrease in peak INa amplitude compared to wild type (WT), whereas under acidotic conditions (pH 7.0), D841H decreased further to 43% along with significant negative shift in inactivation compared to WT at pH 7.4. In induced pluripotent stem-cell-derived cardiomyocytes, similar effects of D841H on INa were observed. VCL colocalized with SCN5A at the intercalated disk in human cardiomyocytes. VCL was also confirmed to directly interact with SCN5A, and VCL-D841H did not disrupt the association of VCL and SCN5A.
CONCLUSIONS: A VCL common variant was genetically and biophysically associated with Chinese SUNDS. The aggravation of loss of function of SCN5A caused by VCL-D841H under acidosis supports that nocturnal sleep respiratory disorders with acidosis may play a key role in the pathogenesis of SUNDS.