Biophysical Characterization of Two Nav1.4 Mutations Identified in Patients with Cold-Induced Myotonia and Periodic Paralysis

Abstract

Mutations in SCN4A gene encoding Nav1.4, the skeletal muscle voltage-gated Na channel underlie several skeletal muscle ion channelopathies whose two major phenotypes include enhanced excitability (myotonia), transient loss of excitability (periodic paralysis), a fluctuation between these two conditions or severe loss of function (myasthenia).Here, we report two novel dominant SCN4A missense mutations: R1451C, found in a patient with paramyotonia congenita (PMC) exhibiting cold-induced myotonia, and R1451L found in a patient diagnosed with periodic paralysis. These mutations are located in the transmembrane segment S4 of DIV domain.To elucidate the mechanism underlying the phenotypes caused by R1451C and R1451L, we used the whole-cell patch-clamp technique to study tsA201 cells expressing WT, R1451C or R1451L channels.Our results show that both mutations impaired fast inactivation kinetics at room temperature, with R1451L being the most affected. However, R1451C exhibited a unique temperature-dependent alteration of the fast inactivation kinetics that can explain the occurrence of myotonia in cold environment. Slow inactivation was assessed for both mutants, and revealed that the recovery from slow inactivation was only slowed for R1451L. This could explain the periodic paralysis phenotype of the patient. Homology modeling and in silico mutagenesis of R1451C and R1451L in the resting state based on a previous model of the channel was also used to probe the potential structural differences between these two mutations. The model revealed a reorganization of the hydrogen bonds between S4 and other segments in both mutations.We conclude from our investigation that the nature of the mutation is accountable for the clinical differences revealed in the patient carriers.