Synthetic peptides corresponding to the four P regions of Electrophorus electricus Na+ channel: interaction with and organization in model phospholipid membranes.

The hydropathy plot of the alpha subunit of the voltage-gated Na+ channel reveals four homologous repeats, each of which is homologous to Shaker type K+ channel monomer and contains six putative transmembrane segments and a hydrophobic segment within the loop connecting transmembrane segments S5 and S6. Current models predict that the four homologous segments [designated H5 or P regions (PR)] from the S5-S6 loop of each repeat lie in the aqueous pore. Peptides corresponding to the P regions of the four domains of the Electrophorus electricus (eel) Na+ channel (25-36 aa long, designated as PR-I, PR-II, PR-III, and PR-IV) and a 23-mer preceding PR-II (designated pre-PR-II) were synthesized and fluorescently labeled. The segments were then structurally and functionally characterized for their interaction with phospholipid membranes. Although the sequences of the four P regions are significantly different, they all bind to zwitterionic phospholipid membranes with similar partition coefficients (approximately 10(4) M-1). The pre-PR-II does not bind membranes at all. Resonance energy transfer measurements, between donor/acceptor-labeled pairs of peptides, revealed that besides the PR-I/PR-III pair, all other pairs form heteroaggregates but do not coassemble with unrelated membrane-bound peptide. Circular dichroism (CD) spectroscopy revealed that PR-I, PR-II, and PR-III adopt similar partial alpha-helical structures (approximately 30%) in 40% trifluoroethanol and in solutions of 1% sodium dodecylsulfate (SDS). The PR-IV (36 aa) adopts approximately 18% alpha-helical structure, and pre-PR-II gives a low CD signal. These findings are in line with proposed models in which the P regions are packed in close proximity in the lumen of the hydrophobic core of the channel. Furthermore, the finding that the PRs adopt similar partial alpha-helical structures in two different hydrophobic environments might suggest that partial alpha-helical structures also exist in the native channel as proposed by recent models. The results are discussed in terms of proposals that various regions of membrane proteins participate in driving folding or oligomerization of the parent molecules.