Sodium channel fragments: Contributions to voltage sensitivity and ion selectivity

The peptide strategy was employed to resolve structure-function relationshi ps in the voltage-dependent sodium channel. Two families of motifs were stu died: the four voltage sensors S4 extended with the short cytoplasmic linke rs L45 and the four P-regions, between S5 and S6, each from the homologous domains of the electric eel sodium channel. Macroscopic conductance experim ents conducted with synthetic S4L45s in neutral lipid planar bilayers point ed to a moderate voltage-sensitivity for repeat IV which has no proline, wh ereas S4L45 of repeats I and II (Pro 19) and especially of repeat III (Pro 14) were much more voltage-sensitive. The influence both of Pro and its pos ition within the sequence was confirmed by comparing the human skeletal mus cle channel isoform D4/S4 wild-type and the R4P analogue. Circular dichrois m spectroscopy shows highest and lowest helicities for repeats IV and III. The conformational transition (from helix to extended, mainly beta forms), which occurs when the solvent dielectric constant increases, was broader wi th repeat III. These structural and functional correlates suggest alternati ve gating mechanisms. The different contributions of each repeat also have effects at the level of the main selectivity filter, which suggests self-re cognition between the four P-regions is a key component of intact sodium ch annel selectivity. In addition, the P-region from domain III is significant ly voltage-sensitive and molecular dynamics simulations show that the C-ter minal part of P-regions is mainly helical whilst the N-terminus tends to un fold. Such specializations of the four domains both in gating and selectivi ty are independently confirmed in recent electrophysiological studies.