Molecular principles of assembly, activation, and inhibition in epithelial sodium channel

The molecular bases of heteromeric assembly and link between Na+ self-inhibition and protease-sensitivity in epithelial sodium channels (ENaCs) are not fully understood. Previously, we demonstrated that ENaC subunits - alpha, beta, and gamma- assemble in a counterclockwise configuration when viewed from outside the cell with the protease-sensitive GRIP domains in the periphery (Noreng et al., 2018). Here we describe the structure of ENaC resolved by cryo-electron microscopy at 3 angstrom. We find that a combination of precise domain arrangement and complementary hydrogen bonding network defines the subunit arrangement. Furthermore, we determined that the a subunit has a primary functional module consisting of the finger and GRIP domains. The module is bifurcated by the alpha 2 helix dividing two distinct regulatory sites: Na+ and the inhibitory peptide. Removal of the inhibitory peptide perturbs the Na+ site via the alpha 2 helix highlighting the critical role of the alpha 2 helix in regulating ENaC function.