Molecular Link Between Voltage-Sensor Modification and Local Anesthetic Block

Abstract

Sodium channels are a major target for many toxins and drugs including local anesthetics (LA). Gating current (Sheets and Hanck, J. Gen. Physiol.; 121(2), 2003) and fluorescence measurements (Muroi and Chanda, J. Gen. Physiol.; 133(1), 2009) show that LA binding to the pore mainly stabilizes the voltage-sensor of domains III of sodium channel in an upward (activated) conformation. The half maximal (V1/2) of the fluorescence-voltage (F-V) curves of probes attached to the voltage-sensor of domain III are left shifted by as much as 50 mV upon LA binding. To address the molecular basis of stabilization of the activated S4 upon LA binding, we systematically introduced tryptophan (or alanine) residues in the S4-S5 linker, N-terminal of S5 and C-terminal of S6 of the domain III muscle sodium channel. We examined the effect of these substitutions by voltage-clamp fluorimetry and by gating current measurements. Mutations of specific residues on the S4-S5 linker and C-terminal of the S6 segment significantly reduced or eliminated the shifts in the F-V curve upon LA binding. Furthermore, the total gating charge in the presence of LA remained unchanged in these mutants. These findings may provide insight into the structure and interactions of intermediate states during the sodium channel gating process.Support: National Institutes of Health, AHA and Shaw Scientific Award