Deubiquitylation Regulates Activation and Proteolytic Cleavage of ENaC

Abstract

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 Ͼ20-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.

Intracellular Ubiquitylation of the Epithelial Na+ Channel Controls Extracellular Proteolytic Channel Activation via Conformational Change

“…Consistently, the balance of ubiquitylation/deubiquitylation (i.e. the ratio of Nedd4-2 to Usp2-45 activity) controls the cleavage of ␣-and ␥-ENaC via 2 different mechanisms (20): 1) Deubiquitylation directly promotes ␣ENaC proteolysis independently of its turnover at the cell surface, whereas 2) ␥ENaC cleavage can be induced by inhibition of endocytosis with dominant-negative dynamin, indicating that prolonged length of time at the cell surface stimulates proteolysis.…”

“…DNA Constructs-All ENaC constructs were based on rat ENaC and most of them described previously (20). Briefly, ␣ENaC-(HA) 3 was subcloned into pCMV4 (21), ␤ENaC into pCDNA3, and ␥ENaC into pcDNA3.1(ϩ)Zeo.…”

“…Furin sites on ␣ENaC (R205A, R231A) or ␥ENaC (R138A) were mutated either in wild-type ENaC or in the lysine mutant. Usp2-45 WT and the inactive mutant (C67A) were generated by PCR as described before and cloned into pcDNA3 (20). The dynamin constructs were previously described (20).…”

“…Anti-Usp2-45 antibodies were used as described before at a dilution of 1:500 (20). Anti-actin (Sigma) is used at a dilution of 1:2,000.…”

“…State-Previous findings suggest that ubiquitylation and deubiquitylation of ENaC (involving Nedd4-2 and/or Usp2-45) affect the proteolytic cleavage of ␣-and ␥-ENaC (19,20). To obtain additional evidence that it is indeed the ubiquitylation of ENaC, which affects the proteolytic cleavage, we expressed in Hek293 cells either wild-type ENaC, or a previously described ENaC mutant (22), in which all cytoplasmic lysines of the 3 subunits were mutated to arginine (ENaC-KR), hence representing a channel complex that cannot become modified by ubiquitin on these sites.…”