The skeletal muscle type voltage-gated sodium channel Nav1.4 plays a crucial role in generating action potentials in myofibers. For many years, gain-of-function variants in the SCN4A gene, which encodes Nav1.4, have been associated with autosomal dominant inherited skeletal muscle channelopathies such as paramyotonia congenita and hypokalemic periodic paralysis. In addition, several loss-of-function variants in SCN4A have been identified in congenital myopathy and neonatal deaths. Recently, compound heterozygous variants in SCN4A , which result in amino acid changes in Nav1.4 (E1266D and F1447S), were reported in a family with neonatal deaths (Hadjipanteli, A., et al. Genes , 2024). In this study, we investigated the function of E1266D- and F1447S-Nav1.4 variants in a heterologous expression system to better understand their pathogenic contribution. First, we quantitatively assessed the membrane expression efficiency of E1266D- and F1447S-Nav1.4 variants in HEK293T cells. Our in-house Cy3-based fluorometric assay revealed that both E1266D- and F1447S-Nav1.4 variants target the plasma membrane as well as wild-type (WT). We then carried out electrophysiological analyses of HEK293T cells singly expressing each Nav1.4 variant. The conductance-voltage relationship of E1266D-Nav1.4 tended to shift toward depolarized potentials, while that of F1447S-Nav1.4 shifted toward hyperpolarized potentials compared to WT. Notably, the steady-state fast inactivation of F1447S-Nav1.4 was shifted by approximately 20 mV toward hyperpolarization relative to WT, which likely confers a loss-of-function effect. F1447 is located immediately before the first gating charge of domain IV S4 crucial to the voltage-sensing operation, and the F to S missense change at this position seems to render the channel dysfunctional. Our results provide evidence of pathological mechanisms associated with neonatal deaths in SCN4A variants. This study was partly supported by AMED (JP24ek0109701) and JSPS (JP25K02581) to T.K., NIH (DC017482) to K.H. and MHLW (23FC1014a) to M.P.T.
Mizobata et al. (Sun,) studied this question.