Molecular Determinants of Coupling Between the Domain III Voltage-sensor and Local Anesthetic Binding Site in the Skeletal Sodium Channel

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, Biophysical Society Meeting, 2008) show that LA binding to the pore mainly stabilizes the voltage-sensor of domains III of sodium channel in an activated conformation. The midpoints 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. How does the binding of LA to the pore affect the movement of the S4-domain III voltage-sensor? To identify the molecular determinants of interaction between the voltage-sensor and LA binding site, we systematically substituted tryptophan residues in the S4-S5 linker and S6 of domain III of skeletal Na+ channel and examined the effect of these substitutions on the movement of DIII-voltage sensor in the absence and presence of LA by voltage-clamp fluorometry technique. A number of mutations in the S4-S5 linker and S6 either showed a significantly diminished or no shift in the F-V curve upon LA binding, suggesting that those residues are involved in coupling the voltage sensor and LA binding site of the sodium channel.Support: National Institutes of Health, AHA and Shaw Scientific Award.