Defining an inhibitory domain in the gamma subunit of the epithelial sodium channel

• Published in: American Journal of Physiology - Renal Physiology 2010-10, Vol.299 (4), p.F854-F861
• Main Authors: Passero, Christopher J, Carattino, Marcelo D, Kashlan, Ossama B, Myerburg, Mike M, Hughey, Rebecca P, Kleyman, Thomas R
• Format: Article
• Language: English
• Subjects: Amino acids; Animals; Binding sites; Cell Line; Cells; Cells, Cultured; Epithelial Sodium Channels - analysis; Epithelial Sodium Channels - drug effects; Epithelial Sodium Channels - metabolism; Female; Frogs; Furin - pharmacology; Gene expression; Humans; Kidney Tubules, Collecting - cytology; Kidney Tubules, Collecting - metabolism; Kidneys; Mice; Oocytes - cytology; Oocytes - metabolism; Peptides; Proteases; Protein Structure, Tertiary; Respiratory Mucosa - cytology; Respiratory Mucosa - metabolism; Serine Endopeptidases - pharmacology; Sodium; Xenopus laevis
• ISSN: 1931-857X; 0363-6127; 1522-1466
• DOI: 10.1152/ajprenal.00316.2010

Proteases activate the epithelial sodium channel (ENaC) by cleaving the large extracellular domains of the α- and γ-subunits and releasing peptides with inhibitory properties. Furin and prostasin activate mouse ENaC by cleaving the γ-subunit at sites flanking a 43 residue inhibitory tract (γE144-K186). To determine whether there is a minimal inhibitory region within this 43 residue tract, we generated serial deletions in the inhibitory tract of the γ-subunit in channels resistant to cleavage by furin and prostasin. We found that partial or complete deletion of a short segment in the γ-subunit, R158-N171, enhanced channel activity. Synthetic peptides overlapping this segment in the γ-subunit further identified a key 11-mer tract, R158-F168 (RFLNLIPLLVF), which inhibited wild-type ENaC expressed in Xenopus laevis oocytes, and endogenous channels in mpkCCD cells and human airway epithelia. Further studies with amino acid-substituted peptides defined residues that are required for inhibition in this key 11-mer tract. The presence of the native γ inhibitory tract in ENaC weakened the intrinsic binding constant of the 11-mer peptide inhibitor, suggesting that the γ inhibitory tract and the 11-mer peptide interact at overlapping sites within the channel.