title: The epithelial Na+ channel is inhibited by a peptide derived from proteolytic processing of its alpha subunit.
abstract: Epithelial sodium channels (ENaCs) mediate Na(+) entry across the apical membrane of high resistance epithelia that line the distal nephron, airway and alveoli, and distal colon. These channels are composed of three homologous subunits, termed alpha, beta, and gamma, which have intracellular amino and carboxyl termini and two membrane-spanning domains connected by large extracellular loops. Maturation of ENaC subunits involves furin-dependent cleavage of the extracellular loops at two sites within the alpha subunit and at a single site within the gamma subunit. The alpha subunits must be cleaved twice, immediately following Arg-205 and Arg-231, in order for channels to be fully active. Channels lacking alpha subunit cleavage are inactive with a very low open probability. In contrast, channels lacking both alpha subunit cleavage and the tract alphaAsp-206-Arg-231 are active when expressed in oocytes, suggesting that alphaAsp-206-Arg-231 functions as an inhibitor that stabilizes the channel in the closed conformation. A synthetic 26-mer peptide (alpha-26), corresponding to alphaAsp-206-Arg-231, reversibly inhibits wild-type mouse ENaCs expressed in Xenopus oocytes, as well as endogenous Na(+) channels expressed in either a mouse collecting duct cell line or primary cultures of human airway epithelial cells. The IC(50) for amiloride block of ENaC was not affected by the presence of alpha-26, indicating that alpha-26 does not bind to or interact with the amiloride binding site. Substitution of Arg residues within alpha-26 with Glu, or substitution of Pro residues with Ala, significantly reduced the efficacy of alpha-26. The peptide inhibits ENaC by reducing channel open probability. Our results suggest that proteolysis of the alpha subunit activates ENaC by disassociating an inhibitory domain (alphaAsp-206-Arg-231) from its effector site within the channel complex.
authors.affiliation: Renal-Electrolyte Division, Department of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania 15261, USA.
authors.forename: Marcelo D
authors.initials: MD
authors.lastname: Carattino
authors.forename: Shaohu
authors.initials: S
authors.lastname: Sheng
authors.forename: James B
authors.initials: JB
authors.lastname: Bruns
authors.forename: Joseph M
authors.initials: JM
authors.lastname: Pilewski
authors.forename: Rebecca P
authors.initials: RP
authors.lastname: Hughey
authors.forename: Thomas R
authors.initials: TR
authors.lastname: Kleyman
mesh_terms: D000818:Animals; D001665:Binding Sites; D004594:Electrophysiology; D053503:Epithelial Sodium Channels; D005260:Female; D015640:Ion Channel Gating / Q000187:drug effects; D051379:Mice; D010446:Peptide Fragments / Q000494:pharmacology; D011485:Protein Binding; D021122:Protein Subunits / Q000737:chemistry*; D015222:Sodium Channels / Q000737:chemistry* / Q000378:metabolism; D013329:Structure-Activity Relationship; D014981:Xenopus
chemical_list: D053503:Epithelial Sodium Channels; D010446:Peptide Fragments; D021122:Protein Subunits; D015222:Sodium Channels