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.

Regulation of the epithelial Na+ channel by paraoxonase-2

“…Like many other membrane-spanning proteins, this trimeric channel complex inefficiently assembles and folds in the ER, and thus only a small fraction of newly synthesized ENaC subunits exit the ER as assembled channels and traffic to the plasma membrane (27)(28)(29). Indeed, several molecular chaperones have been implicated in key steps during ENaC biogenesis, including subunit folding and channel assembly within the ER (30,31). Other factors regulate intracellular trafficking, plasma membrane insertion, and retrieval as well as both ER-associated and lysosome-mediated degradation of ENaC (32)(33)(34)(35)(36)(37).…”

Paraoxonase 3 functions as a chaperone to decrease functional expression of the epithelial sodium channel

“…It is possible that PON3 facilitates proteasomal degradation of misfolded ENaC subunits. Other chaperones have been identified that affect ENaC trafficking at later sites in the secretory pathway (40,41,43,44,103). Additional studies are needed to investigate mechanisms by which PONs regulate ENaC expression and physiological relevance of this regulation in the kidney and other organs.…”

“…Together, this multi-stage machinery determines the total number of functional channel (N) at the cell surface. Molecular chaperones have been implicated in multiple key steps during ENaC biogenesis, trafficking and degradation (34,(40)(41)(42)(43)(44)(45), including members of the paraoxonase (PON) family.…”

SPLUNC1 is an allosteric modulator of the epithelial sodium channel

“…Of note, when the Ca 2+ ‐sensing protein, stromal interacting protein 1, relocated to the endoplasmic reticulum–plasma membrane junction, we observed a similar (~50%) change in mobility (32). Chaperones have been identified that facilitate ENaC trafficking to the plasma membrane, including Hsp40, Jem1, Scj1, and BiP (41), as well as Hsc70 and ERp29 (42, 43). More recently, it has been shown that β‐ENaC is required to prevent Lhs1‐induced endoplasmic reticulum–associated degradation of α‐ENaC (45).…”