(a) Cartoon representation of ENaC perpendicular to the membrane where the GRIP domains in all three subunits are shown as surface representation. α, β and γ are colored blue, red and magenta, respectively. Additionally, the inhibitory P1 peptides of αGRIP and γGRIP are highlighted in yellow. (b) Overall view of the inhibitory peptide pocket in the α subunit. The finger, thumb, and P3-P4 strands of the GRIP domains are colored purple, green, and blue, respectively, and shown in surface. The inhibitory peptide is shown in sticks representation and colored yellow. In the α subunit, the inhibitory peptide only extends to the −3 position. (c) Close-up view of the pocket consisting of conserved residues Pro187, Leu188, and Trp251. Leu188 makes hydrophobic contacts with Trp251 from the α2 helix and Ile224 of the P3 strand of the GRIP domain. (d) Overall view of the inhibitory peptide pocket in the γ subunit. Representations are similar to (b). In the γ subunit, the inhibitory peptide extends to position −6 making more extensive contacts with the finger and thumb domains. (e) Close-up view of the binding pocket consisting of the equivalent residues shown in (c). In the γ subunit, the Leu160 primarily interacts with the residues in the α2 helix. The residues in the GRIP domain that interact with the inhibitory peptides are in sticks representation in panels (c) and (e). (f) Sequence alignment of ENaC GRIP domains (hENaCα, GenBank ID:4506815; hENaCβ, 124301096; hENaCγ, 42476333). The sequences were aligned with Clustal Omega and manually adjusted. Coloring or shading is as follows: cysteines participating in disulfide bonds are in red boxes, glycosylation sites (predicted and/or present in cryo-EM map) are shown as bold N, furin sites are in green, prostasin site in magenta, and the P1 peptides in α and γ are in blue.