Tryptophan Scanning Mutagenesis to Identify the Residues Involved in Coupling between the Pore and DIII Voltage-Sensor of a Sodium Channel

Abstract

In response to membrane depolarization, voltage-gated ion channels undergo a structural rearrangement that moves the voltage sensing segments in the electric field and initiates a series of conformational transitions that ultimately opens the channel pore. The mechanism of coupling between the voltage-sensing domain and pore domain remains poorly understood. To characterize the molecular basis of this coupling, we have systematically substituted tryptophan residues in the S4-S5 region (from C-terminus of S4 to N-terminus of S5) and C-terminus of S6 of domain III in the skeletal muscle sodium channel. The effects of these perturbations on the movement of the voltage-sensor were monitored by using a site-specific fluorescent reporter on S4 of domain III using voltage-clamp fluorometry. Conformational changes in the pore were tracked by measuring the inward sodium currents. Our study identifies a number of mutants, which stabilize the voltage-sensors in the activated conformation while destabilizing the open pore conformation relative to the wild type. We suggest that the residues at these positions play an important role in coupling the voltage-sensor of domain III to the pore of the sodium channel. Support: National Institutes of Health, American heart Association and Shaw Scientist Award.