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The Epithelial Na+ Channel Is Inhibited by a Peptide Derived from Proteolytic Processing of Its α Subunit

J. Biol. Chem., 7 July 2006
Vol. 281, Issue 27, p. 18901-18907
DOI: 10.1074/jbc.M604109200

The Epithelial Na+ Channel Is Inhibited by a Peptide Derived from Proteolytic Processing of Its α Subunit

  1. Marcelo D. Carattino,
  2. Shaohu Sheng,
  3. James B. Bruns,
  4. Joseph M. Pilewski§,
  5. Rebecca P. Hughey,1 and
  6. Thomas R. Kleyman
  1. Renal-Electrolyte Division and §Division of Pulmonary, Allergy, and Critical Care Medicine, Department of Medicine, and Department of Cell Biology and Physiology, University of Pittsburgh, Pittsburgh, Pennsylvania 15261
  1. 1 To whom correspondence should be addressed: Renal-Electrolyte Division, University of Pittsburgh, S933 Scaife Hall, 3550 Terrace St., Pittsburgh, PA 15261. Tel.: 412-383-8949; Fax: 412-383-8956; E-mail: hughey{at}dom.pitt.edu.

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 α, β, and γ, 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 α subunit and at a single site within the γ subunit. The α subunits must be cleaved twice, immediately following Arg-205 and Arg-231, in order for channels to be fully active. Channels lacking α subunit cleavage are inactive with a very low open probability. In contrast, channels lacking both α subunit cleavage and the tract αAsp-206-Arg-231 are active when expressed in oocytes, suggesting that αAsp-206-Arg-231 functions as an inhibitor that stabilizes the channel in the closed conformation. A synthetic 26-mer peptide (α-26), corresponding to αAsp-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 IC50 for amiloride block of ENaC was not affected by the presence of α-26, indicating that α-26 does not bind to or interact with the amiloride binding site. Substitution of Arg residues within α-26 with Glu, or substitution of Pro residues with Ala, significantly reduced the efficacy of α-26. The peptide inhibits ENaC by reducing channel open probability. Our results suggest that proteolysis of the α subunit activates ENaC by disassociating an inhibitory domain (αAsp-206-Arg-231) from its effector site within the channel complex.

  • Received May 1, 2006.

Citation:

M. D. Carattino, S. Sheng, J. B. Bruns, J. M. Pilewski, R. P. Hughey, and T. R. Kleyman, The Epithelial Na+ Channel Is Inhibited by a Peptide Derived from Proteolytic Processing of Its α Subunit. J. Biol. Chem. 281, 18901-18907 (2006).

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