An Inhibitory Peptide Derived from the α-subunit of the Epithelial Sodium Channel (ENaC) Shows a Helical Conformation

Proteolytic activation of the heteromeric epithelial sodium channel (ENaC) is thought to involve the release of inhibitory peptides from the extracellular domains of its alpha- and gamma- subunit. Recently, we demonstrated that an alpha-13-mer peptide, corresponding to a putative inhibitory region within the extracellular domain of human alpha ENaC, inhibits human alpha beta gamma ENaC. The aim of the present study was to investigate the structural basis of the inhibitory effect of this alpha-13-mer peptide. Analysis of the peptide by replica exchange molecular dynamics method, circular dichroism spectroscopy, nuclear magnetic resonance spectroscopy, and molecular dynamics simulations suggested that a helical turn at the carboxy-terminus is the preferred conformational state of the alpha-13-mer peptide. From this we predicted that a specific mutation (leucine 188 to alanine) should have a strong effect on the conformational preferences of the peptide. To functionally test this, we compared the effect of the wild-type alpha-13-mer with that of a mutant alpha-L188A-13-mer on ENaC currents in Xenopus laevis oocytes heterologously expressing human alpha beta gamma ENaC. We demonstrated that replacing the leucine 188 by alanine abolished the inhibitory effect of the alpha-13-mer peptide on ENaC. These findings suggest that a helical conformation in its carboxyterminal part is functionally important to mediate ENaC inhibition by the alpha-13-mer peptide. However, high resolution structural information on the complex of the inhibitory alpha ENaC peptide and the channel are needed to confirm this conclusion. Copyright (c) 2012 S. Karger AG, Basel