A Structural Rearrangement in the Sodium Channel Pore Linked to Slow Inactivation and Use Dependence

Voltage-gated sodium (Na⁺) channels are a fundamental target for modulating excitability in neuronal and muscle cells. When depolarized, Na⁺ channels may gradually enter long-lived, slow-inactivated conformational states, causing a cumulative loss of function. Although the structural motifs that underlie transient, depolarization-induced Na⁺ channel conformational states are increasingly recognized, the conformational changes responsible for more sustained forms of inactivation are unresolved. Recent studies have shown that slow inactivation components exhibiting a range of kinetic behavior (from tens of milliseconds to seconds) are modified by mutations in the outer pore P-segments. We examined the state-dependent accessibility of an engineered cysteine in the domain III, P-segment (F1236C; rat skeletal muscle) to methanethiosulfonate-ethylammonium (MTSEA) using whole-cell current recordings in HEK 293 cells. F1236C was reactive with MTSEA applied from outside, but not inside the cell, and modification was markedly increased by depolarization. Depolarized F1236C channels exhibited both intermediate (I_M; τ ∼ 30 ms) and slower (I_S; τ ∼ 2 s) kinetic components of slow inactivation. Trains of brief, 5-ms depolarizations, which did not induce slow inactivation, produced more rapid modification than did longer (100 ms or 6 s) pulse widths, suggesting both the I_M and I_S kinetic components inhibit depolarization-induced MTSEA accessibility of the cysteine side chain. Lidocaine inhibited the depolarization-dependent sulfhydryl modification induced by sustained (100 ms) depolarizations, but not by brief (5 ms) depolarizations. We conclude that competing forces influence the depolarization-dependent modification of the cysteine side chain: conformational changes associated with brief periods of depolarization enhance accessibility, whereas slow inactivation tends to inhibit the side chain accessibility. The findings suggest that slow Na⁺ channel inactivation and use-dependent lidocaine action are linked to a structural rearrangement in the outer pore.