Diverse biophysical mechanisms in voltage-gated sodium channel Nav1.4 variants associated with myotonia

Mutations in SCN4A gene encoding Nav1.4 channel alpha-subunit, are known to cause neuromuscular disorders such as myotonia or paralysis. Here, we study the effect of two amino acid replacements, K1302Q and G1306E, in the DIII-IV loop of the channel, corresponding to mutations found in patients with myotonia. We combine clinical, electrophysiological, and molecular modeling data to provide a holistic picture of the molecular mechanisms operating in mutant channels and eventually leading to pathology. We analyze the existing clinical data for patients with the K1302Q substitution, which was reported for adults with or without myotonia phenotypes, and report two new unrelated patients with the G1306E substitution, who presented with severe neonatal episodic laryngospasm and childhood-onset myotonia. We provide a functional analysis of the mutant channels by expressing Nav1.4 alpha-subunit in Xenopus oocytes in combination with beta 1 subunit and recording sodium currents using two-electrode voltage clamp. The K1302Q variant exhibits abnormal voltage dependence of steady-state fast inactivation, being the likely cause of pathology. K1302Q does not lead to decelerated fast inactivation, unlike several other myotonic mutations such as G1306E. For both mutants, we observe increased window currents corresponding to a larger population of channels available for activation. To elaborate the structural rationale for our experimental data, we explore the contacts involving K/Q1302 and E1306 in the AlphaFold2 model of wild-type Nav1.4 and Monte Carlo-minimized models of mutant channels. Our data provide the missing evidence to support the classification of K1302Q variant as likely pathogenic and may be used by clinicians. Mutations in SCN4A gene encoding the skeletal muscle-type voltage-gated sodium channel Nav1.4 alpha-subunit can lead to myotonia. Replacements in the DIII-DIV linker lead to significantly different biophysical alterations, and yet the resulting clinical condition is similar. In K1302Q, we observe a depolarizing shift in steady-state inactivation, whereas in K1302R there is a hyperpolarizing shift of the voltage dependence of activation, and in G1306E the two effects occur simultaneously. In all cases, the so-called "window" current is enhanced.image