Epithelial sodium channel exit from the endoplasmic reticulum is regulated by a signal within the carboxyl cytoplasmic domain of the alpha subunit.
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.
Mueller
Gunhild M
GM
Renal-Electrolyte Division, Department of Medicine.
Kashlan
Ossama B
OB
Renal-Electrolyte Division, Department of Medicine.
Bruns
James B
JB
Renal-Electrolyte Division, Department of Medicine.
Maarouf
Ahmad B
AB
Renal-Electrolyte Division, Department of Medicine.
Aridor
Meir
M
Department of Cell Biology and Physiology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261.
Kleyman
Thomas R
TR
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.
Hughey
Rebecca P
RP
Renal-Electrolyte Division, Department of Medicine; Department of Cell Biology and Physiology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261.
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
D053503:Epithelial Sodium Channels; D005723:gamma-Glutamyltransferase; D006026:Glycoside Hydrolases