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 α, β, and γ 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 α and γ subunits. To identify signals in ENaC for ER retention/retrieval or ER exit/release, chimera were prepared with the interleukin α subunit (Tac) and each of the three cytoplasmic carboxyl termini of mouse ENaC (Tac-Ct) or with γ-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-αCt when compared with Tac. ER exit of ENaC was assayed after metabolic labeling by following the appearance of cleaved α as cleaved α subunit, but not non-cleaved α, is endoglycosidase H-resistant. Interestingly ER exit of epitope-tagged and truncated α (αΔ624–699-V5) with full-length βγ was similar to wild type α (+βγ), whereas ER exit of ENaC lacking the entire cytoplasmic carboxyl tail of α (αΔ613–699-V5 +βγ) was significantly reduced. Subsequent analysis of ER exit for ENaCs with mutations within the intervening sequence 613HRFRSRYWSPG623 within the context of the full-length α revealed that mutation αRSRYW620 to AAAAA significantly reduced ER exit. These data indicate that ER exit of ENaC is regulated by a signal within the α subunit carboxyl cytoplasmic tail.