Defining an inhibitory domain in the gamma subunit of the epithelial sodium channel.
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.
Passero
Christopher J
CJ
Renal Electrolyte Div., Dept. of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania 15261, USA.
Carattino
Marcelo D
MD
Kashlan
Ossama B
OB
Myerburg
Mike M
MM
Hughey
Rebecca P
RP
Kleyman
Thomas R
TR
D000818:Animals; D002460:Cell Line; D002478:Cells, Cultured; D053503:Epithelial Sodium Channels / Q000032:analysis* / Q000187:drug effects / Q000378:metabolism; D005260:Female; D045683:Furin / Q000494:pharmacology; D006801:Humans; D007685:Kidney Tubules, Collecting / Q000166:cytology / Q000378:metabolism; D051379:Mice; D009865:Oocytes / Q000166:cytology / Q000378:metabolism; D017434:Protein Structure, Tertiary; D020545:Respiratory Mucosa / Q000166:cytology / Q000378:metabolism; D012697:Serine Endopeptidases / Q000494:pharmacology; D014982:Xenopus laevis; D000099271:Prostasin
D053503:Epithelial Sodium Channels; D045683:Furin; D012697:Serine Endopeptidases; D000099271:Prostasin