Phosphatidylinositol 4-phosphate 5-kinase reduces cell surface expression of the epithelial sodium channel (ENaC) in cultured collecting duct cells

Kelly M. Weixel, Robert S. Edinger, Lauren Kester, Christopher J. Guerriero, Huamin Wang, Liang Fang, Thomas R. Kleyman, Paul A. Welling, Ora A. Weisz, John P. Johnson

Research output: Contribution to journalArticle

Abstract

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 α (PI5KIα) has been shown to enhance endocytosis in a PIP2-dependent manner. We tested the hypothesis that PI5KIα-mediated PIP2 production would negatively regulate ENaC current by enhancing epsin-mediated endocytosis of the channel. Expression of PI5KIα decreased ENaC currents in Xenopus oocytes by 80%, entirely because of a decrease in cell surface ENaC levels. Catalytically inactive mutants of PI5Kα had no effect on ENaC activity. Expression of the PIP2 binding region of epsin increased ENaC current in oocytes, an effect completely reversed by coexpression of PI5KIα. Overexpression of epsin reduced amiloride-sensitive current in CCD cells. Overexpression of PI5KIα enhanced membrane PIP2 levels and reduced apical surface expression of ENaC in CCD cells, down-regulating amiloride-sensitive current. Knockdown of PI5KIα with isoform-specific siRNA resulted in a 4-fold enhancement of ENaC activity. PI5KIα 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 PI5KIα.

Original languageEnglish (US)
Pages (from-to)36534-36542
Number of pages9
JournalJournal of Biological Chemistry
Volume282
Issue number50
DOIs
Publication statusPublished - Dec 14 2007
Externally publishedYes

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ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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