Defining an inhibitory domain in the [alpha]-subunit of the epithelial sodium channel

Epithelial sodium channels (ENaC) are processed by proteases as they transit the biosynthetic pathway. We recently observed that furin-dependent processing of the [alpha]-subunit of ENaC at two sites within its extracellular domain is required for channel activation due to release of a 26-residue inhibitory domain. While channels with [alpha]-subunits lacking the furin sites are not cleaved and have very low activity, channels lacking the furin consensus sites as well as the tract between these sites ([alpha]D206-R231) are active. We analyzed channels with a series of deletions in the tract [alpha]D206-R231 and lacking the [alpha]-subunit furin consensus sites in Xenopus laevis oocytes. We found an eight-residue tract that, when deleted, restored channel activity to the level found in oocytes expressing wild-type ENaC. A synthetic peptide, LPHPLQRL, representing the tract [alpha]L211-L218, inhibited wild-type ENaC expressed in oocytes with an [IC.sub.50] of 0.9 [micro]M, and inhibited channels expressed in collecting duct cells and human primary airway epithelial cells with an [IC.sub.50]s of between ~50 and 100 [micro]M. Analyses of peptides with deletions within this inhibitory tract indicate that eight residues is the minimal backbone length that is required for ENaC inhibition. Analyses of 8-mer peptides with conserved and nonconserved substitutions suggest that [L.sup.1], [P.sup.2], [H.sup.3], [P.sup.4], and [L.sup.8] are required for inhibitory activity. Our findings suggest that this eight-residue tract is a key conserved inhibitory domain that provides epithelial cells with a reserve of inactive channels that can be activated as required by proteases. peptide inhibitors; proteases; furin