title: The deubiquitinating enzyme UCH-L3 regulates the apical membrane recycling of the epithelial sodium channel.
abstract: The epithelial sodium channel (ENaC) is ubiquitinated by the E3 ligase Nedd4-2 at the apical membranes of polarized cortical collecting duct (CCD) epithelial cells. This leads to ENaC endocytosis and possible degradation. Because ENaC is known to recycle at the apical membranes of CCD cells, deubiquitinating enzymes (DUBs) are likely involved in regulating ENaC surface density by facilitating ENaC recycling as opposed to degradation. Using a chemical probe approach to tag active DUBs, we identified ubiquitin C-terminal hydrolase (UCH) isoform L3 as the predominant DUB in endosomal compartments of CCD cells. Blocking UCH-L3 activity or reducing its expression by selective knockdown increased ENaC ubiquitination and resulted in its removal from the apical membranes of CCD cells. Functionally this caused a rapid reduction in transepithelial Na(+) currents across the CCD epithelia. Surface biotinylation demonstrated the loss of ENaC from the apical surface when UCH-L3 was inhibited. Whole cell or apical surface immunoprecipitation demonstrated increased ENaC ubiquitination with UCH-L3 inhibition. This constitutes a novel function for UCH in epithelia and in the regulation of ion channels and demonstrates the dynamic regulation of apically located ENaC by recycling, which is facilitated by this DUB.
authors.affiliation: Department of Cell Biology and Physiology, School of Medicine, University of Pittsburgh, Pittsburgh, PA 15261, USA. michael7@pitt.edu
authors.forename: Michael B
authors.initials: MB
authors.lastname: Butterworth
authors.forename: Robert S
authors.initials: RS
authors.lastname: Edinger
authors.forename: Huib
authors.initials: H
authors.lastname: Ovaa
authors.forename: Danny
authors.initials: D
authors.lastname: Burg
authors.forename: John P
authors.initials: JP
authors.lastname: Johnson
authors.forename: Raymond A
authors.initials: RA
authors.lastname: Frizzell
mesh_terms: D000818:Animals; D019921:Biotinylation; D002462:Cell Membrane / Q000378:metabolism; D002966:Clathrin / Q000737:chemistry; D003546:Cysteine Endopeptidases / Q000378:metabolism / Q000502:physiology*; D011992:Endosomes / Q000378:metabolism; D004847:Epithelial Cells; D053503:Epithelial Sodium Channels / Q000378:metabolism*; D007685:Kidney Tubules, Collecting / Q000378:metabolism; D051379:Mice; D008954:Models, Biological; D020033:Protein Isoforms; D017434:Protein Structure, Tertiary; D034741:RNA, Small Interfering / Q000378:metabolism; D013997:Time Factors; D025801:Ubiquitin / Q000737:chemistry*; D043222:Ubiquitin Thiolesterase / Q000378:metabolism / Q000502:physiology*
chemical_list: D002966:Clathrin; D053503:Epithelial Sodium Channels; D020033:Protein Isoforms; D034741:RNA, Small Interfering; C526917:UCHL3 protein, mouse; D025801:Ubiquitin; D043222:Ubiquitin Thiolesterase; D003546:Cysteine Endopeptidases