The epithelial Na+ channel subunit autoinhibitory tract suppresses channel activity by binding the γ subunit's finger-thumb domain interface

Epithelial Na+ channel (ENaC) maturation and activation require proteolysis of both the and subunits. Cleavage at multiple sites in the finger domain of each subunit liberates their autoinhibitory tracts. Synthetic peptides derived from the proteolytically released fragments inhibit the channel, likely by reconstituting key interactions removed by the proteolysis. We previously showed that a peptide derived from the subunit's autoinhibitory sequence (-8) binds at the subunit's finger-thumb domain interface. Despite low sequence similarity between the and subunit finger domains, we hypothesized that a peptide derived from the subunit's autoinhibitory sequence (-11) inhibits the channel through an analogous mechanism. Using Xenopus oocytes, we found here that channels lacking a subunit thumb domain were no longer sensitive to -11, but remained sensitive to -8. We identified finger domain sites in the subunit that dramatically reduced -11 inhibition. Using cysteines and sulfhydryl reactive cross-linkers introduced into both the peptide and the subunit, we also could cross-link -11 to both the finger domain and the thumb domain of the subunit. Our results suggest that -8 and -11 occupy similar binding pockets within their respective subunits, and that proteolysis of the and subunits activate the channel through analogous mechanisms.