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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., (Copyright © 2014 the American Physiological Society.)

The epithelial sodium channel (ENaC) plays an important role in homeostasis of blood pressure and of the airway surface liquid, and excess function of ENaC results in refractory hypertension (in Liddle's syndrome) and impaired mucociliary clearance (in cystic fibrosis). The regulation of ENaC by molecular chaperones, such as the 70-kDa heat shock protein Hsc70, is not completely understood. Our previously published data suggest that Hsc70 negatively affects ENaC activity and surface expression in Xenopus oocytes; here we investigate the mechanism by which Hsc70 acts on ENaC in epithelial cells. In Madin-Darby canine kidney cells stably expressing epitope-tagged αβγ-ENaC and with tetracycline-inducible overexpression of Hsc70, treatment with 5 μg/ml doxycycline increased total Hsc70 expression 20%. This increase in Hsc70 expression led to a decrease in ENaC activity and surface expression that corresponded to an increased rate of functional ENaC retrieval from the cell surface. In addition, Hsc70 overexpression decreased the association of newly synthesized ENaC subunits. These data support the hypothesis that Hsc70 inhibits ENaC functional expression at the apical surface of epithelia by regulating ENaC biogenesis and ENaC trafficking at the cell surface.