Asymmetric functional contributions of acidic and aromatic side chains in sodium channel voltage-sensor domains

Voltage-gated sodium (Na(V)) channels mediate electrical excitability in animals. Despite strong sequence conservation among the voltage-sensor domains (VSDs) of closely related voltage-gated potassium (K(V)) and Na(V) channels, the functional contributions of individual side chains in Na(v) VSDs remain largely enigmatic. To this end, natural and unnatural side chain substitutions were made in the S2 hydrophobic core (HC), the extracellular negative charge cluster (ENC), and the intracellular negative charge cluster (INC) of the four VSDs of the skeletal muscle sodium channel isoform (Na(V)1.4). The results show that the highly conserved aromatic side chain constituting the S2 HC makes distinct functional contributions in each of the four Na(V) domains. No obvious cation–pi interaction exists with nearby S4 charges in any domain, and natural and unnatural mutations at these aromatic sites produce functional phenotypes that are different from those observed previously in K(v) VSDs. In contrast, and similar to results obtained with K(v) channels, individually neutralizing acidic side chains with synthetic derivatives and with natural amino acid substitutions in the INC had little or no effect on the voltage dependence of activation in any of the four domains. Interestingly, countercharge was found to play an important functional role in the ENC of DI and DII, but not DIII and DIV. These results suggest that electrostatic interactions with S4 gating charges are unlikely in the INC and only relevant in the ENC of DI and DII. Collectively, our data highlight domain-specific functional contributions of highly conserved side chains in Na(V) VSDs.