Abstract
Epithelial Na+ channel (ENaC) maturation and activation require proteolysis of both the α and γ 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 α subunit's autoinhibitory sequence (α-8) binds at the α subunit's finger-thumb domain interface. Despite low sequence similarity between the α and γ subunit finger domains, we hypothesized that a peptide derived from the γ subunit's autoinhibitory sequence (γ-11) inhibits the channel through an analogous mechanism. Using Xenopus oocytes, we found here that channels lacking a γ subunit thumb domain were no longer sensitive to γ-11, but remained sensitive to α-8. We identified finger domain sites in the γ subunit that dramatically reduced γ-11 inhibition. Using cysteines and sulfhydryl reactive cross-linkers introduced into both the peptide and the subunit, we also could cross-link γ-11 to both the finger domain and the thumb domain of the γ subunit. Our results suggest that α-8 and γ-11 occupy similar binding pockets within their respective subunits, and that proteolysis of the α and γ subunits activate the channel through analogous mechanisms.
Highlights
Epithelial Na؉ channel (ENaC) maturation and activation require proteolysis of both the ␣ and ␥ subunits
We previously reported that ␣-8 inhibits ENaC currents by binding at the ␣ subunit’s finger–thumb domain interface (Fig. 1) [12, 14]
Consistent with competition between the autoinhibitory tract in the ␥ subunit and ␥-11, we previously demonstrated that channels that retain the ␥ subunit autoinhibitory tract are more poorly inhibited by ␥-11 than channels that lack the ␥ subunit autoinhibitory tract [13]
Summary
Cleaves the ␣ subunit both proximal and distal to the ␣-autoinhibitory tract, following RXXR motifs [4]. Proteases regulate ENaC in the lung airway and alveoli [7], where ENaC regulates the airway surface liquid height [8]. In these and other tissues, proteolysis of the ␥ subunit at the cell surface may play important physiologic roles or contribute to the pathophysiology of disease. We previously reported that a peptide derived from the ␣-autoinhibitory tract (␣-8, Fig. 1B) inhibits the channel by binding the finger–thumb domain interface of the ␣ subunit [12]. We show that the ␥ subunit thumb domain is required for ␥-11 inhibition, but not for ␣-8 inhibition This suggests independent binding sites for ␣-8 and ␥-11. Our data suggest that cleavage of the ␣ and ␥ subunits activates ENaC through analogous mechanisms, i.e. by removing autoinhibitory domains from the finger–thumb interfaces of the respective subunits
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