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J. Biol. Chem., Vol. 282, Issue 50, 36534-36542, December 14, 2007

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Phosphatidylinositol 4-Phosphate 5-Kinase Reduces Cell Surface Expression of the Epithelial Sodium Channel (ENaC) in Cultured Collecting Duct Cells^*

Kelly M. Weixel¹, Robert S. Edinger, Lauren Kester, Christopher J. Guerriero², Huamin Wang, Liang Fang, Thomas R. Kleyman, Paul A. Welling, Ora A. Weisz, and John P. Johnson³

From the Department of Medicine, Renal and Electrolyte Division, University of Pittsburgh, Pittsburgh, Pennsylvania 15261 and the Department of Physiology, University of Maryland School of Medicine, Baltimore, Maryland 21201

Ubiquitination of ENaC subunits has been shown to negatively regulate the cell surface expression of ENaC channels. We have previously demonstrated that epsin links ubiquitinated ENaC to clathrin adaptors for clathrin-mediated endocytosis. Epsin is thought to directly modify the curvature of membranes upon binding to phosphatidylinositol 4,5-bisphosphate (PIP₂) where it recruits clathrin and stimulates lattice assembly. Murine phosphatidylinositol 4-phosphate 5-kinase (PI5KI) has been shown to enhance endocytosis in a PIP₂-dependent manner. We tested the hypothesis that PI5KI-mediated PIP₂ production would negatively regulate ENaC current by enhancing epsin-mediated endocytosis of the channel. Expression of PI5KI decreased ENaC currents in Xenopus oocytes by 80%, entirely because of a decrease in cell surface ENaC levels. Catalytically inactive mutants of PI5K had no effect on ENaC activity. Expression of the PIP₂ binding region of epsin increased ENaC current in oocytes, an effect completely reversed by co-expression of PI5KI. Overexpression of epsin reduced amiloride-sensitive current in CCD cells. Overexpression of PI5KI enhanced membrane PIP₂ levels and reduced apical surface expression of ENaC in CCD cells, down-regulating amiloride-sensitive current. Knockdown of PI5KI with isoform-specific siRNA resulted in a 4-fold enhancement of ENaC activity. PI5KI localized exclusively to the apical plasma membrane domain when overexpressed in mouse CCD cells, consistent for a role in regulating PIP₂ production at the apical plasma membrane. We conclude that membrane turnover events regulating ENaC surface expression and activity in oocytes and CCD cells can be regulated by PI5KI.

Received for publication, May 14, 2007 , and in revised form, October 11, 2007.

^* This work was supported in part by Grants DK 57718, DK47874 (to J. P. J.), DK 064613 (to O. A. W.), DK 65161 (to T. S. K.), DK-63049 and DK-54231 (to P. A. W.) from the National Institutes of Health (NIH). 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 Supported in part by an NIH Institutional National Research Service Award T32 DK61296.

² Supported in part by an NIH Institutional National Research Service Award T32 DK61296 and American Heart Association predoctoral fellowship.

³ To whom correspondence should be addressed: Dept. of Medicine, Renal and Electrolyte Division, University of Pittsburgh, 3500 Terrace St., Pittsburgh, PA 15261. Tel.: 412-648-9075; Fax: 412-383-8956; E-mail: Johnson{at}dom.pitt.edu.

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