Mechanistic Diversity and NaV1.5 Failure Modes in SUNDS-Associated SCN5A Variants: Integrating Gating Physiology with Structural Modeling

Mechanistic diversity and NaV1.5 failure modes in SUNDS-associated SCN5A variants: Integrating gating physiology with structural modeling Background: Sodium channel dysfunction is a central mechanism underlying inherited arrhythmia syndromes that cause sudden death during sleep, yet the mechanistic spectrum of SCN5A variants implicated in sudden unexplained nocturnal death syndrome (SUNDS) remains poorly defined. Although clinical cohorts have reported functional data for select SCN5A variants, no mechanistic studies have examined variants identified directly from forensic SUNDS cases, particularly in Southeast Asia, the region with the highest incidence. This gap limits our ability to link NaV1.5 gating defects to arrhythmogenic mechanisms in SUNDS. Objective: To define the electrophysiological and structural mechanisms by which SUNDS-associated SCN5A variants disrupt NaV1.5 function and to develop a mechanistic failure-mode framework applicable to postmortem variant interpretation. Hypothesis: SUNDS victims carry pathogenic SCN5A variants that perturb NaV1.5 activation and/or inactivation through distinct biophysical mechanisms, including loss of function, gain of function, or mixed dysfunction, each increasing vulnerability to nocturnal ventricular arrhythmias. Methods: Genomic data from 110 SUNDS cases from a high-incidence Southeast Asian cohort were reviewed. Novel missense variants were generated by site-directed mutagenesis and expressed in HEK293 cells for whole-cell patch-clamp analysis (n = 15 per group). Structural modeling was performed to predict how each variant perturbs local residue networks that are likely to disrupt channel function. The combined electrophysiology-structural workflow was designed as a translational platform for variant interrogation. Results: Eleven victims carried SCN5A variants; four cases with novel and missense variants were functionally characterized. All deaths occurred during sleep with normal autopsy findings. Failure mode 1 - Loss of function (Brugada-like): p.A665S significantly reduced peak INa (p = 0.012). Failure mode 2 - Gain of function (Long QT syndrome type 3 LQTS3-like): p.R179Q accelerated recovery from inactivation (p = 0.011). Failure mode 3 - Mixed severe dysfunction (overlap phenotype): p.E171G and p.G599R markedly suppressed peak INa (p = 0.0001; p < 0.0001). p.N1659S produced profound peak-INa loss together with an approximately 100-fold increase in sustained INa (p < 0.0001). Structural modeling revealed disruption of the stabilizing N1659-Q1491 electrostatic interaction, compromising the inactivation "lid" and explaining the combined gain- and loss-of-function profile. Variants with no detectable functional alteration: p.A178T and p.Q998K exhibited normal NaV1.5 gating parameters. Conclusion: This study establishes the first mechanistic classification of SUNDS-associated SCN5A variants and demonstrates distinct NaV1.5 failure modes that unify Brugada-, LQTS3-, and overlap-like behaviors. By integrating patch-clamp biophysics with structural modeling, we provide a transferable framework for precision autopsy, variant interpretation, and mechanistic discovery. These findings advance physiological understanding of NaV1.5 dysfunction and illustrate an emerging investigator-driven platform capable of resolving complex genotype-to-phenotype relationships in inherited arrhythmia syndromes. Funding: Health Systems Research Institute (Grant No. 66-082); National Research Council of Thailand (NRCT) Mid-Career Researcher Grant; Development and Promotion of Science and Technology Talents Project Fund. This abstract was presented at the American Physiology Summit 2026 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.