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, cau sing a cumulative loss of function. Although the structural motifs that und erlie transient, depolarization-induced Na+ channel conformational states a re 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 (f rom tens of milliseconds to seconds) are modified by mutations in the outer pore P-segments. We examined the state-dependent accessibility of an engin eered cysteine in the domain III, P-segment (F1236C; rat skeletal muscle) t o methanethiosulfonate-ethylammonium (MTSEA) using whole-cell current recor dings in HEK 293 cells. F1236C was reactive with MTSEA applied from outside , but not inside the cell, and modification was markedly increased by depol arization. Depolarized F1236C channels exhibited both intermediate (I-M; ta u similar to 30 ms) and slower (I-S; tau similar to 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 comp onents inhibit depolarization-induced MTSEA accessibility of the cysteine s ide chain. Lidocaine inhibited the depolarization-dependent sulfhydryl modi fication induced by sustained (100 ms) depolarizations, but not by brief (5 ms) depolarizations. We conclude that competing forces influence the depol arization-dependent modification of the cysteine side chain: conformational changes associated with brief periods of depolarization enhance accessibil ity, whereas slow inactivation tends to inhibit the side chain accessibilit y. The findings suggest that slow Na+ channel inactivation and use-dependen t lidocaine action are linked to a structural rearrangement in the outer po re.