Mapping an inhibitory peptide binding site to construct a molecular model of the alpha subunit of the epithelial sodium channel

Abstract

The epithelial Na+ channel (ENaC) facilitates transepithelial Na+ transport in several tissues to regulate extracellular fluid volume and airway surface liquid volume. The channel is assembled from three homologous subunits with the bulk of each protein's mass found in the extracellular domains. We previously showed that two of these subunits are cleaved at multiple sites, resulting in channel activation due to the release of inhibitory tracts. Synthetic peptides based on these tracts inhibit the channel. We sought to define the binding site for the alpha‐subunit derived peptide by systematically mutating large stretches of ENaC and assaying for channel inhibition. Here we present molecular models constructed using distance based restraints derived from our data in conjunction with restraints derived from the closely related protein acid sensing ion channel 1. Our data show that the inhibitory peptide binds to the so‐called finger and thumb domains of ENaC. Our models suggest that the interface between the thumb and finger domains is critical to the control of channel gating.