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J. Biol. Chem., Vol. 282, Issue 52, 37885-37893, December 28, 2007

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The Deubiquitinating Enzyme UCH-L3 Regulates the Apical Membrane Recycling of the Epithelial Sodium Channel^*

Michael B. Butterworth¹, Robert S. Edinger, Huib Ovaa^¶, Danny Burg^¶, John P. Johnson, and Raymond A. Frizzell

From the Department of Cell Biology and Physiology and the Renal Electrolyte Division, School of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania 15261 and the ^¶Netherlands Cancer Institute, Division of Cellular Biochemistry, 1066 CX Amsterdam, The Netherlands

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.

Received for publication, September 24, 2007 , and in revised form, October 16, 2007.

^* This work was supported by the Cystic Fibrosis Foundation Grant BUTTER06G0 (to M. B. B.), National Institutes of Health Grants DK57718 and DK47874 (to J. P. J.) and DK54814 (to R. A. F.), and a VIDI grant from the Netherlands Foundation for Scientific Research (to H. O.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Fig. S1.

¹ To whom correspondence should be addressed: Dept. of Cell Biology & Physiology, University of Pittsburgh, S375 BST, 3500 Terrace St., Pittsburgh, PA 15261. Tel.: 412-383-8591; Fax: 412-648-8330; E-mail: michael7{at}pitt.edu.

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