Effects of Charged Residues Inserted above R1 in S4-Based Voltage Sensors

Abstract

Membrane proteins containing S4-based voltage sensors domains (VSD) respond to changes in the membrane potential by transferring electrically charged side chains (gating charges), mainly located in the S4 segment, across the membrane electric field. Yet, in the archetypical Shaker Kv channel, not all positive S4 residues contribute to the gating charge. Moreover, the number and positions of charged residues near the external side of S4 is poorly conserved amongst VSDs, suggesting that some S4 charged residues may have a regulatory role. We investigated this hypothesis by individually inserting Arg residues at the top of the S4 segment of the Shaker channel, from positions 356 to 361, and determining the impact of the mutations on the channel's gating currents. Our results show a non-ambiguous periodicity of the phenotypes that correlate with the periodicity of the S4 alpha-helical structure: Arg inserted on the same side of the gating charge positively-shifted the charge vs. voltage (Q-V) curve, while Arg inserted on the opposite side negatively-shifted the Q-V curve. We propose two distinct mechanisms that account for these observations. Charged side chains pointing towards the gating charge pathway exert a local bias in the electric field sensed by the innermost native Arg. However, charged side chains located on the opposite side of the gating charge pathway tend to favor more depolarized conformations of the S4 helix to avoid being in proximity to the hydrophobic environment of the membrane lipids. We propose that naturally-occurring charged residues located at the top of S4 may help to finely tune the VSD biophysical properties. This mechanism is illustrated by a myasthenia-associated muscle sodium channel variant that introduced the V1442E mutation at the top of the S4 in domain IV. This work was supported by NIH grant GM030376.