title: ERp29 regulates epithelial sodium channel functional expression by promoting channel cleavage.
abstract: The epithelial Na(+) channel (ENaC) plays a key role in the regulation of blood pressure and airway surface liquid volume. ERp29 is a 29-kDa thioredoxin-homologous endoplasmic reticulum (ER) protein that has only a single cysteine instead of the usual thioredoxin CXXC motif. Our group previously demonstrated that ERp29 promotes biogenesis of the cystic fibrosis transmembrane conductance regulator (CFTR). On the basis of similarities of CFTR and ENaC trafficking, we hypothesized that ERp29 would also regulate ENaC biogenesis and functional expression. In epithelial cells, overexpression of wild-type (wt) ERp29 increased ENaC functional expression [amiloride-sensitive short-circuit current (Isc)] in Ussing chamber experiments, as well as the abundance of the cleaved form of γ-ENaC in whole cell lysates. In contrast, siRNA-mediated depletion of ERp29 or overexpression of a mutant ERp29 lacking its single cysteine (C157S ERp29) decreased ENaC functional expression. Cells in which wt ERp29 was overexpressed had a smaller fractional increase in amiloride-sensitive Isc when trypsin was applied to the apical surface to activate uncleaved ENaC, while cells in which C157S ERp29 was overexpressed or ERp29 was depleted had a significantly greater fractional increase in amiloride-sensitive Isc in response to trypsin. Interestingly, these observations were not associated with altered expression of β-ENaC at the apical surface. Instead, ERp29 appeared to promote the interaction of β-ENaC with the Sec24D cargo recognition component of the coat complex II ER exit machinery. Together, these data support the hypothesis that ERp29 directs ENaC toward the Golgi, where it undergoes cleavage during its biogenesis and trafficking to the apical membrane.
authors.affiliation: Division of Pulmonary Medicine and Cystic Fibrosis Center, Children's Hospital of Philadelphia, Philadelphia, Pennsylvania; and.
authors.forename: Yael
authors.initials: Y
authors.lastname: Grumbach
authors.affiliation: Division of Pulmonary Medicine and Cystic Fibrosis Center, Children's Hospital of Philadelphia, Philadelphia, Pennsylvania; and.
authors.forename: Yann
authors.initials: Y
authors.lastname: Bikard
authors.affiliation: Division of Pulmonary Medicine and Cystic Fibrosis Center, Children's Hospital of Philadelphia, Philadelphia, Pennsylvania; and.
authors.forename: Laurence
authors.initials: L
authors.lastname: Suaud
authors.affiliation: Division of Pulmonary Medicine and Cystic Fibrosis Center, Children's Hospital of Philadelphia, Philadelphia, Pennsylvania; and.
authors.forename: Rebecca A
authors.initials: RA
authors.lastname: Chanoux
authors.affiliation: Division of Pulmonary Medicine and Cystic Fibrosis Center, Children's Hospital of Philadelphia, Philadelphia, Pennsylvania; and Department of Pediatrics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania rrubenst@mail.med.upenn.edu.
authors.forename: Ronald C
authors.initials: RC
authors.lastname: Rubenstein
mesh_terms: D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D004285:Dogs; D053503:Epithelial Sodium Channels / Q000378:metabolism*; D015870:Gene Expression; D006360:Heat-Shock Proteins / Q000502:physiology*; D006801:Humans; D061985:Madin Darby Canine Kidney Cells; D008564:Membrane Potentials; D051379:Mice; D011499:Protein Processing, Post-Translational; D021381:Protein Transport; D059748:Proteolysis; D033921:Vesicular Transport Proteins / Q000378:metabolism; D014982:Xenopus laevis
chemical_list: C491738:ERP29 protein, human; D053503:Epithelial Sodium Channels; D006360:Heat-Shock Proteins; C530641:SEC24D protein, human; D033921:Vesicular Transport Proteins
keywords: biogenesis; chaperone; endoplasmic reticulum; epithelium; trafficking