title: Epithelial sodium channel exit from the endoplasmic reticulum is regulated by a signal within the carboxyl cytoplasmic domain of the alpha subunit.
abstract: Epithelial sodium channels (ENaCs) are assembled in the endoplasmic reticulum (ER) from alpha, beta, and gamma 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 alpha and gamma subunits. To identify signals in ENaC for ER retention/retrieval or ER exit/release, chimera were prepared with the interleukin alpha subunit (Tac) and each of the three cytoplasmic carboxyl termini of mouse ENaC (Tac-Ct) or with gamma-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-alphaCt when compared with Tac. ER exit of ENaC was assayed after metabolic labeling by following the appearance of cleaved alpha as cleaved alpha subunit, but not non-cleaved alpha, is endoglycosidase H-resistant. Interestingly ER exit of epitope-tagged and truncated alpha (alphaDelta624-699-V5) with full-length betagamma was similar to wild type alpha (+betagamma), whereas ER exit of ENaC lacking the entire cytoplasmic carboxyl tail of alpha (alphaDelta613-699-V5 +betagamma) 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 alpha revealed that mutation alphaRSRYW(620) to AAAAA significantly reduced ER exit. These data indicate that ER exit of ENaC is regulated by a signal within the alpha subunit carboxyl cytoplasmic tail.
authors.affiliation: Renal-Electrolyte Division, Department of Medicine.
authors.forename: Gunhild M
authors.initials: GM
authors.lastname: Mueller
authors.affiliation: Renal-Electrolyte Division, Department of Medicine.
authors.forename: Ossama B
authors.initials: OB
authors.lastname: Kashlan
authors.affiliation: Renal-Electrolyte Division, Department of Medicine.
authors.forename: James B
authors.initials: JB
authors.lastname: Bruns
authors.affiliation: Renal-Electrolyte Division, Department of Medicine.
authors.forename: Ahmad B
authors.initials: AB
authors.lastname: Maarouf
authors.affiliation: Department of Cell Biology and Physiology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261.
authors.forename: Meir
authors.initials: M
authors.lastname: Aridor
authors.affiliation: Renal-Electrolyte Division, Department of Medicine; Department of Cell Biology and Physiology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261. Electronic address: kleyman@pitt.edu.
authors.forename: Thomas R
authors.initials: TR
authors.lastname: Kleyman
authors.affiliation: Renal-Electrolyte Division, Department of Medicine; Department of Cell Biology and Physiology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261.
authors.forename: Rebecca P
authors.initials: RP
authors.lastname: Hughey
mesh_terms: D000818:Animals; D002460:Cell Line; D003593:Cytoplasm / Q000378:metabolism; D004285:Dogs; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D053503:Epithelial Sodium Channels / Q000378:metabolism*; D006026:Glycoside Hydrolases / Q000378:metabolism; D006056:Golgi Apparatus / Q000378:metabolism; D051379:Mice; D008954:Models, Biological; D009154:Mutation; D017434:Protein Structure, Tertiary; D013997:Time Factors; D005723:gamma-Glutamyltransferase / Q000378:metabolism
chemical_list: D053503:Epithelial Sodium Channels; D005723:gamma-Glutamyltransferase; D006026:Glycoside Hydrolases