Integrated electrophysiological, cellular, and pharmacological profiling reveals variant-specific mechanisms in SCN4A-related myotonia

Non-dystrophic myotonias are rare skeletal muscle channelopathies characterized by delayed muscle relaxation and clinical stiffness. This study investigates the molecular mechanisms underlying three missense variants in SCN4A encoding for the voltage gated skeletal muscle sodium channel NaV1.4-p.K1308R, p.R1451H, and p.M1701V-identified in patients with non-dystrophic myotonia. All probands carried these variants in combination with the ClC-1 p.G190S mutation, a digenic configuration likely contributing to the heterogeneous clinical manifestations. An integrated approach combining electrophysiology, protein trafficking assessment, and evaluation of endoplasmic reticulum stress, revealed distinct pathogenic signatures for each variant. p.K1308R and p.M1701V primarily altered channel gating, whereas p.R1451H exhibited profound cellular impairment, including intracellular retention, reduced membrane expression, and robust activation of endoplasmic reticulum-stress pathways. Functionally, p.K1308R and p.R1451H caused marked reductions in current density and slowed activation kinetics, while p.M1701V produced milder perturbations consistent with its generally less severe phenotype. Because cold exposure exacerbated symptoms in carriers of p.R1451H and p.M1701V, the functional behaviour of these variants was examined at 37 °C. Both showed recovery of current amplitude to wild-type values and normalization of activation voltage dependence, although inactivation defects persisted. These temperature-dependent improvements were accompanied by increased window current probability, indicating partial temperature-dependent stabilization of channel gating. Pharmacological testing revealed that mexiletine modulates gating abnormalities in a variant-specific manner, reinforcing the clinical relevance of mechanistic stratification. Overall, the findings showed that clinically similar myotonic phenotypes may arise from divergent molecular defects and emphasize the relevance of precision medicine approaches tailored to variant-specific pathogenic mechanisms.