Epithelial Sodium Channel Exit from the Endoplasmic Reticulum Is Regulated by a Signal within the Carboxyl Cytoplasmic Domain of the α Subunit

Abstract

Epithelial sodium channels (ENaCs) are assembled in the endoplasmic reticulum (ER) from ␣, ␤, and ␥ subunits, each with two transmembrane domains, a large extracellular loop, and cytoplasmic amino and carboxyl termini. ENaC maturation involves transit through the Golgi complex where Asn-linked glycans are processed to complex type and the channel is activated by furin-dependent cleavage of the ␣ and ␥ subunits. To identify signals in ENaC for ER retention/retrieval or ER exit/ release, chimera were prepared with the interleukin ␣ subunit (Tac) and each of the three cytoplasmic carboxyl termini of mouse ENaC (Tac-Ct) or with ␥-glutamyltranspeptidase and each of the three cytoplasmic amino termini (Nt-GGT). By monitoring acquisition of endoglycosidase H resistance after metabolic labeling, we found no evidence of ER retention of any chimera when compared with control Tac or GGT, but we did observe enhanced exit of Tac-␣Ct when compared with Tac. ER exit of ENaC was assayed after metabolic labeling by following the appearance of cleaved ␣ as cleaved ␣ subunit, but not noncleaved ␣, is endoglycosidase H-resistant. Interestingly ER exit of epitope-tagged and truncated ␣ (␣⌬624 -699-V5) with fulllength ␤␥ was similar to wild type ␣ (؉␤␥), whereas ER exit of ENaC lacking the entire cytoplasmic carboxyl tail of ␣ (␣⌬613-699-V5 ؉␤␥) was significantly reduced. Subsequent analysis of ER exit for ENaCs with mutations within the intervening sequence 613 HRFRSRYWSPG 623 within the context of the full-length ␣ revealed that mutation ␣RSRYW 620 to AAAAA significantly reduced ER exit. These data indicate that ER exit of ENaC is regulated by a signal within the ␣ subunit carboxyl cytoplasmic tail.

SPLUNC1 regulates airway surface liquid volume by protecting ENaC from proteolytic cleavage

“…Proteolytic cleavage of ␣ and ␥ subunits is required for ENaC activation (21,22), and because CAP2 is highly expressed in human bronchial epithelial cultures (11), we tested whether SPLUNC1 could alter ␣ and ␥ ENaC cleavage by CAP2. Due to the relative scarcity of purified rSPLUNC1, we elected to coexpress SPLUNC1 and ENaC, rather than use rSPLUNC1 for subsequent oocyte studies, because SPLUNC1 is secreted at sufficient quantities from oocytes to inhibit ENaC (Fig.…”

Cys Palmitoylation of the β Subunit Modulates Gating of the Epithelial Sodium Channel

“…7) that regulates ENaC endocytosis and recycling at the cell surface (63). For the same reason, it is unlikely that ␤ subunit palmitoylation affects coat protein complex II binding to ␣RSRYW620 and thereby ER exit (7).…”

“…Assembly is inefficient, and the majority of newly synthesized subunits undergo ER-associated degradation with a half-life of 1-2 h as determined by metabolic labeling (3)(4)(5)(6)(7). A small pool of newly synthesized subunits has a significantly longer half-life (Ͼ4 h) and represents assembled channels that have exited the ER and are present in later compartments.…”

“…This latter pool contains subunits whose N-glycans have undergone both Golgi-dependent terminal processing and cleavage of ␣ and ␥ by furin, a protease localized primarily to the trans-Golgi network (5,8,9). ENaC exit from the ER is regulated by a signal within the C-terminal cytoplasmic domain of the ␣ subunit (␣RSRYW620 in murine ENaC) that resembles the well described cytoplasmic dibasic motifs recognized by the coat protein complex II prebudding (7). Interestingly, some fraction of ENaC reaches the cell surface lacking Golgi-associated processing: the N-glycans are not processed, and the ␣ and ␥ subunits are not cleaved (11).…”

Hsc70 negatively regulates epithelial sodium channel trafficking at multiple sites in epithelial cells

“…Either the initial assembly of ENaC subunits or their exit from the ER appears to be inefficient in model systems, and a significant fraction of the newly synthesized channel is targeted for degradation by an ER-associated degradation (ERAD) pathway (35,60,64). The COOH terminus of ␣-ENaC appears to control ENaC's exit from the ER and contains a consensus sequence for interaction with coat complex II (COP II) machinery (44). In fact, our group's recent data present evidence that ENaC does, in fact, interact with COP II and that promotion of this interaction by overexpression of Hsp70, the stressinduced 70-kDa heat shock protein, is associated with increased ENaC activity (11).…”