Role of Nedd4-2 and polyubiquitination in epithelial sodium channel degradation in untransfected renal A6 cells expressing endogenous ENaC subunits.
Amiloride-sensitive epithelial sodium channels (ENaC) are responsible for transepithelial Na(+) transport in the kidney, lung, and colon. The channel consists of three subunits (alpha, beta, and gamma). In Madin-Darby canine kidney (MDCK) cells and Xenopus laevis oocytes transfected with all three ENaC subunits, neural precursor cell-expressed developmentally downregulated protein (Nedd4-2) promotes ubiquitin conjugation of ENaC. For native proteins in some cells, ubiquitin conjugation is a signal for their degradation by the ubiquitin-proteasome pathway, whereas in other cell types ubiquitin conjugation is a signal for endocytosis and lysosomal protein degradation. When ENaC are transfected into MDCK cells, ubiquitin conjugation leads to lysosomal degradation. In this paper, we characterize the involvement of the ubiquitin-proteasome proteolytic pathway in the regulation of functional ENaC in untransfected renal A6 cells expressing native ENaC subunits. In contrast to transfected cells, we show that total cellular alpha-, beta-, and gamma-ENaC subunits are polyubiquitinated and that ubiquitin conjugation of subunits increases when the cells are treated with a proteasome inhibitor. We show that Nedd4-2 is associated with alpha- and beta-subunits and is associated with the apical membrane. We also show the Nedd4-2 can regulate the number of functional ENaC subunits in the apical membrane. The results reported here suggest that the ubiquitin-proteasome proteolytic pathway is an important determinant of ENaC function in untransfected renal cells expressing endogenous ENaC.
Malik
B
B
Dept. of Physiology, Emory University School of Medicine, Atlanta, GA 30322, USA. bmalik@physio.emory.edu
Yue
Q
Q
Yue
G
G
Chen
X J
XJ
Price
S R
SR
Mitch
W E
WE
Eaton
D C
DC
D000818:Animals; D002460:Cell Line; D056827:Endosomal Sorting Complexes Required for Transport; D004847:Epithelial Cells / Q000378:metabolism; D053503:Epithelial Sodium Channels; D007668:Kidney / Q000166:cytology / Q000378:metabolism*; D000075702:Nedd4 Ubiquitin Protein Ligases; D025821:Polyubiquitin / Q000502:physiology*; D015222:Sodium Channels / Q000378:metabolism*; D044767:Ubiquitin-Protein Ligases / Q000502:physiology*; D029867:Xenopus Proteins; D014982:Xenopus laevis
D056827:Endosomal Sorting Complexes Required for Transport; D053503:Epithelial Sodium Channels; D015222:Sodium Channels; D029867:Xenopus Proteins; D025821:Polyubiquitin; D000075702:Nedd4 Ubiquitin Protein Ligases; C000618482:Nedd4 protein, Xenopus; C000618460:nedd4l protein, Xenopus; D044767:Ubiquitin-Protein Ligases