Contributions of Conserved Negative Surface Potentials in S2 and S3 to Voltage-Gating of Sodium Channels

The question of how S4 charges are stabilized in the hydrophobic membrane environment is of critical importance to understand the function of voltage-gated ion channels (VGICs). Besides lipids, proteinaceous interactions are thought to play an important role and, indeed, the electronegative surface potentials of aromatic side chains and the net negative charge of acidic side chains are well suited to fulfill this role. Interestingly, a single aromatic side chain in S2 and a number of acidic side chains in S2 and S3 are highly conserved among VGICs. These residues have been extensively studied in potassium channels but little is known about their role in sodium channels. Here we incorporate unnatural amino acids to probe their contributions to channel gating in the skeletal sodium channel isoform (Nav1.4). Replacing the S2 aromatic in domains I-IV with fluorinated Phe derivatives does not affect channel gating, ruling out a cation-pi interaction with S4 charges. Trp substitutions in this position lead to a significant right-shift in the GV in domain I only, highlighting not only the functional differences between Nav domains, but also sodium and potassium channels, as the latter show a dramatic left-shift in the GV when Trp is introduced at the equivalent position in S2. Furthermore, we replaced Glu side chains with the neutral isosteric Glu analog nitrohomolalanine (Nha). Our data show that neutralizing a negative charge in S2 near the extracellular end of S4 leads to a significant right-shift in the GV in domain I, while neutralizing negative charges in S2 and S3 near the intracellular end of S4 result in small or no shifts in the GV. Together, these results shed new light on our understanding of voltage-gating in sodium channels.