A critical role for the branched sidechain adjacent to the third arginine of the sodium channel voltage sensor

Molecular dissection of the action potential sodium channel with the peptide strategy pleads for functional and structural asymmetry. The authors previously showed that the different voltage dependencies exhibited by the four isolated S4L45 (from the electric eel sequence) reconstituted into planar lipid bilayers were correlated with the position of a single proline residue and to a conformational transition (from helix to extended forms) occurring with an increase of the solvent permittivity. Ferroelechic liquid crystal properties of residues in an α helix predict a loss of ferroelectric behavior when hydrophobic residues with branched side chains are replaced by residues with unbranched side chains. To test the influence of unbranched side chains, the residues Ile and Leu next to the third and fifth arginines respectively in repeat III were replaced either with α-methylalanine or alanine. The modified voltage sensors were assayed for the voltage sensitivity of their macroscopic conductances, their secondary structure and stability and behavior in analytical ultracentrifugation. Whereas the α-methylalanine analog retained a high sensitivity to voltage changes, but induced longer single-channel events, the alanine substitution (next to the third arginine, R10) leads to a loss of this intrinsic property of the native repeat III, which was correlated with a tendency for dimerization. These results suggest a role for branched side chains of specific residues involved in gating, in agreement with the ferroelectric liquid crystal model