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Deubiquitylation Regulates Activation and Proteolytic Cleavage of ENaC

Dorothée Ruffieux-Daidié^*, Olivier Poirot^*, Sheerazed Boulkroun^*, François Verrey, Stephan Kellenberger^* and Olivier Staub^*

^* Department of Pharmacology and Toxicology, University of Lausanne, Lausanne, and Institute of Physiology, University of Zurich, Zurich, Switzerland

Correspondence: Dr. Olivier Staub, Department of Pharmacology and Toxicology, University of Lausanne, Rue du Bugnon 27, CH-1005 Lausanne, Switzerland. Phone: +41-21-692-5407; Fax: +41-21-692-5355; E-mail: olivier.staub{at}unil.ch

Received for publication October 23, 2007. Accepted for publication May 28, 2008.

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 ENaC and ENaC at their extracellular loops. When endocytosis was inhibited with dominant negative dynamin (DynK44R), channel density and ENaC cleavage were increased, but ENaC cleavage and ENaC activity were not augmented. When Usp2-45 was coexpressed with DynK44R, both ENaC cleavage and activity were recovered. In summary, these data suggest that Usp2-45 deubiquitylation of ENaC enhances the proteolytic activation of both ENaC and ENaC, possibly by inducing a conformational change and by interfering with endocytosis, respectively.

Copyright © 2008 by the American Society of Nephrology. Online ISSN: 1533-3450 Print ISSN: 1046-6673