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 – a, b, and g – 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 Å. 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 a2 helix dividing two distinct regulatory sites: Na⁺ and the inhibitory peptide. Removal of the inhibitory peptide perturbs the Na⁺ site via the a2 helix highlighting the critical role of the a2 helix in regulating ENaC function.