Vasopressin-inducible ubiquitin-specific protease 10 increases ENaC cell surface expression by deubiquitylating and stabilizing sorting nexin 3.
Adjustment of Na+ balance in extracellular fluids is achieved by regulated Na+ transport involving the amiloride-sensitive epithelial Na+ channel (ENaC) in the distal nephron. In this context, ENaC is controlled by a number of hormones, including vasopressin, which promotes rapid translocation of water and Na+ channels to the plasma membrane and long-term effects on transcription of vasopressin-induced and -reduced transcripts. We have identified a mRNA encoding the deubiquitylating enzyme ubiquitin-specific protease 10 (Usp10), whose expression is increased by vasopressin at both the mRNA and the protein level. Coexpression of Usp10 in ENaC-transfected HEK-293 cells causes a more than fivefold increase in amiloride-sensitive Na+ currents, as measured by whole cell patch clamping. This is accompanied by a three- to fourfold increase in surface expression of alpha- and gamma-ENaC, as shown by cell surface biotinylation experiments. Although ENaC is well known to be regulated by its direct ubiquitylation, Usp10 does not affect the ubiquitylation level of ENaC, suggesting an indirect effect. A two-hybrid screen identified sorting nexin 3 (SNX3) as a novel substrate of Usp10. We show that it is a ubiquitylated protein that is degraded by the proteasome; interaction with Usp10 leads to its deubiquitylation and stabilization. When coexpressed with ENaC, SNX3 increases the channel's cell surface expression, similarly to Usp10. In mCCD(cl1) cells, vasopressin increases SNX3 protein but not mRNA, supporting the idea that the vasopressin-induced Usp10 deubiquitylates and stabilizes endogenous SNX3 and consequently promotes cell surface expression of ENaC.
Boulkroun
Sheerazed
S
Department of Pharmacology and Toxicology, University of Lausanne, Rue du Bugnon 27, CH-1005 Lausanne, Switzerland.
Ruffieux-Daidié
Dorothée
D
Vitagliano
Jean-Jacques
JJ
Poirot
Olivier
O
Charles
Roch-Philippe
RP
Lagnaz
Dagmara
D
Firsov
Dmitri
D
Kellenberger
Stephan
S
Staub
Olivier
O
D000818:Animals; D050034:Antidiuretic Agents / Q000378:metabolism* / Q000494:pharmacology; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D053503:Epithelial Sodium Channels / Q000378:metabolism*; D006801:Humans; D007668:Kidney / Q000166:cytology / Q000502:physiology*; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D051379:Mice; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D012964:Sodium / Q000378:metabolism; D058305:Sorting Nexins; D014162:Transfection; D020798:Two-Hybrid System Techniques; D025801:Ubiquitin / Q000378:metabolism; D043222:Ubiquitin Thiolesterase / Q000235:genetics / Q000378:metabolism*; D014667:Vasopressins / Q000378:metabolism* / Q000494:pharmacology; D033921:Vesicular Transport Proteins / Q000235:genetics / Q000378:metabolism*
D050034:Antidiuretic Agents; D002352:Carrier Proteins; D053503:Epithelial Sodium Channels; D012333:RNA, Messenger; C533230:Snx3 protein, mouse; D058305:Sorting Nexins; D025801:Ubiquitin; D033921:Vesicular Transport Proteins; D014667:Vasopressins; D012964:Sodium; D043222:Ubiquitin Thiolesterase