A leaky inactivated state in Nav1.4 created by mutating hydrophobic residues in DIII, DIV S6 segments.

Abstract

Fast inactivation in Nav channels is essential to shorten the action potential. The canonical model for fast inactivation proposed that the IFM motif in the DIII-DIV linker blocks the channel producing fast inactivation but recent structural studies indicate that large residues in the S6 segments are more likely to act as the fast inactivation gate. Here, we studied the effects of reducing the volume of structurally identified residues in S6 we hypothesize to be part of the inactivation gate. In both DIII and DIV, mutating two large residues to alanine (mutants DIIIAA & DIVAA) creates significant amount of steady state current while none of the single mutations does. In DIII, both individual single mutants significantly shifted the GV curve to the right. However, the GV of the steady current of DIIIAA, showed a shift to the left instead. We hypothesized that by reducing the volumes of the residues in DIII S6, we partially opened the fast inactivation gate, and ions could permeate through the closed inactivated state leading to the apparent left shift of the GV curve. To test this, in the DIIIAA mutant we introduced the IQM mutation, which has been shown to prevent entrance into fast inactivation and as expected, DIIIAA_IQM showed a right-shifted GV curve, similar to the ones seen in single mutations. Finally, we recorded the gating current from DIIIAA and DIVAA, and characterized the charge immobilization. Despite significant steady state current, both DIIIAA and DIVAA showed similar time course and degree of charge immobilization as the wild type. We concluded that by reducing the volumes of the residues, we created a leaky inactivated state in the Nav channels, through which the steady current was conducted. Support NIHR01GM030376.