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J. Biol. Chem., Vol. 282, Issue 52, 37402-37411, December 28, 2007

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The Epithelial Sodium Channel (ENaC) Traffics to Apical Membrane in Lipid Rafts in Mouse Cortical Collecting Duct Cells^*

Warren G. Hill¹, Michael B. Butterworth¹, Huamin Wang^¶, Robert S. Edinger^¶, Jonathan Lebowitz^¶, Kathryn W. Peters, Raymond A. Frizzell, and John P. Johnson^¶2

From the Division of Matrix Biology, Department of Medicine, Harvard Medical School, Beth Israel Deaconess Medical Center, Boston, Massachusetts 02215 and the Department of Cell Biology and Physiology and the ^¶Laboratory of Epithelial Cell Biology, Renal-Electrolyte Division, Department of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania 15261

We previously showed that ENaC is present in lipid rafts in A6 cells, a Xenopus kidney cell line. We now demonstrate that ENaC can be detected in lipid rafts in mouse cortical collecting duct (_MPKCCD₁₄) cells by detergent insolubility, buoyancy on density gradients using two distinct approaches, and colocalization with caveolin 1. Less than 30% of ENaC subunits were found in raft fractions. The channel subunits also colocalized on sucrose gradients with known vesicle targeting and fusion proteins syntaxin 1A, Vamp 2, and SNAP23. Hormonal stimulation of ENaC activity by either forskolin or aldosterone, short or long term, did not alter the lipid raft distribution of ENaC. Methyl-β-cyclodextrin added apically to _MPKCCD₁₄ cells resulted in a slow decline in amiloride-sensitive sodium transport with short circuit current reductions of 38.1 ± 9.6% after 60 min. The slow decline in ENaC activity in response to apical cyclodextrin was identical to the rate of decline seen when protein synthesis was inhibited by cycloheximide. Apical biotinylation of _MPKCCD₁₄ cells confirmed the loss of ENaC at the cell surface following cyclodextrin treatment. Acute stimulation of the recycling pool of ENaC was unaffected by apical cyclodextrin application. Expression of dominant negative caveolin isoforms (CAV1-eGFP and CAV3-DGV) which disrupt caveolae, reduced basal ENaC currents by 72.3 and 78.2%, respectively; but, as with cyclodextrin, the acute response to forskolin was unaffected. We conclude that ENaC is present in and regulated by lipid rafts. The data are consistent with a model in which rafts mediate the constitutive apical delivery of ENaC.

Received for publication, May 17, 2007 , and in revised form, October 4, 2007.

^* This work was supported by Grants DK47874, DK57718 (to J. P. J.), DK43955, DK48217 (to W. G. H.), and DK54814 (to R. F.) from the National Institutes of Health. 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.

¹ Both authors contributed equally to this study.

² To whom correspondence should be addressed: 935 Scaife Hall, Renal-Electrolyte Division, University of Pittsburgh, 3550 Terrace St., Pittsburgh, PA15261. Tel.: 412-648-9075; Fax: 412-383-8956; E-mail: Johnson{at}dom.pitt.edu.

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