Studies of α-Helicity and Intersegmental Interactions in Voltage-Gated Na+ Channels: S2D4

Much data, including crystallographic, support structural models of sodium and potassium channels consisting of S1-S4 transmembrane segments (the "voltage-sensing domain") clustered around a central pore-forming region (S5-S6 segments and the intervening loop). Voltage gated sodium channels have four non-identical domains which differentiates them from the homotetrameric potassium channels that form the basis for current structural models. Since potassium and sodium channels also exhibit many different functional characteristics and the fourth domain (D4) of sodium channels differs in function from other domains (D1-D3), we have explored its structure in order to determine whether segments in D4 of sodium channels differ significantly from that determined for potassium channels. We have probed the secondary and tertiary structure and the role of the individual amino acid residues of the S2D4) of Na(v)1.4 by employing cysteine-scanning mutagenesis (with tryptophan and glutamine substituted for native cysteine). A Fourier transform power spectrum of perturbations in free energy of steady-state inactivation gating (using midpoint potentials and slopes of Boltzmann equation fits of channel availability, h(infinity)-V plots) indicates a substantial amount of alpha-helical structure in S2D4 (peak at 106 degrees, alpha-Periodicity Index (alpha-PI) of 3.10), This conclusion is supported by alpha-PI values of 3.28 and 2.84 for the perturbations in rate constants of entry into (beta) and exit from (alpha) fast inactivation at 0 mV for mutant channels relative to WT channels assuming a simple two-state model for transition from the open to inactivated state. The results of cysteine substitution at the two most sensitive sites of the S2D4 alpha-helix (N1382 and E1392C) support the existence of electrostatic network interactions between S2 and other transmembrane segments within Na(v)1.4D4 similar to but not identical to those proposed for K+ channels.