Deubiquitylation regulates activation and proteolytic cleavage of ENaC.
The epithelial sodium channel (ENaC) is critical for sodium and BP homeostasis. ENaC is regulated by Nedd4-2-mediated ubiquitylation, which leads to its internalization; this process can be reversed by deubiquitylation, which is regulated by the aldosterone-induced enzyme Usp2-45. In a second regulatory pathway, ENaC can be activated by luminal serine protease-mediated cleavage of its extracellular loops. Whether these two regulatory processes interact, however, is unknown. Here, in HEK293 cells stably transfected with ENaC, Usp2-45 interacted with ENaC, leading to deubiquitylation of the channel and stimulation of ENaC activity >20-fold. This was accompanied by a modest increase in cell surface expression of ENaC and by proteolytic cleavage of alphaENaC and gammaENaC at their extracellular loops. When endocytosis was inhibited with dominant negative dynamin (DynK44R), channel density and gammaENaC cleavage were increased, but alphaENaC cleavage and ENaC activity were not augmented. When Usp2-45 was coexpressed with DynK44R, both alphaENaC cleavage and activity were recovered. In summary, these data suggest that Usp2-45 deubiquitylation of ENaC enhances the proteolytic activation of both alphaENaC and gammaENaC, possibly by inducing a conformational change and by interfering with endocytosis, respectively.
Ruffieux-Daidié
Dorothée
D
Department of Pharmacology and Toxicology, University of Lausanne, Rue du Bugnon 27, CH-1005 Lausanne, Switzerland.
Poirot
Olivier
O
Boulkroun
Sheerazed
S
Verrey
François
F
Kellenberger
Stephan
S
Staub
Olivier
O
D000584:Amiloride / Q000494:pharmacology; D000818:Animals; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D010450:Endopeptidases / Q000378:metabolism; D056827:Endosomal Sorting Complexes Required for Transport; D053503:Epithelial Sodium Channels / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D000075702:Nedd4 Ubiquitin Protein Ligases; D010447:Peptide Hydrolases / Q000378:metabolism; D021122:Protein Subunits; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D026941:Sodium Channel Blockers / Q000494:pharmacology; D014162:Transfection; D043222:Ubiquitin Thiolesterase; D044767:Ubiquitin-Protein Ligases / Q000378:metabolism; D054875:Ubiquitination
D056827:Endosomal Sorting Complexes Required for Transport; D053503:Epithelial Sodium Channels; D021122:Protein Subunits; D011994:Recombinant Proteins; D026941:Sodium Channel Blockers; D000584:Amiloride; C000618479:NEDD4L protein, rat; D000075702:Nedd4 Ubiquitin Protein Ligases; C000618483:Nedd4 protein, human; C000618484:Nedd4 protein, rat; C000618485:Nedd4L protein, human; D044767:Ubiquitin-Protein Ligases; D010450:Endopeptidases; D010447:Peptide Hydrolases; C108665:USP2 protein, human; D043222:Ubiquitin Thiolesterase