Phosphatidylinositol 4-phosphate 5-kinase reduces cell surface expression of the epithelial sodium channel (ENaC) in cultured collecting duct cells.
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 alpha (PI5KIalpha) has been shown to enhance endocytosis in a PIP2-dependent manner. We tested the hypothesis that PI5KIalpha-mediated PIP2 production would negatively regulate ENaC current by enhancing epsin-mediated endocytosis of the channel. Expression of PI5KIalpha decreased ENaC currents in Xenopus oocytes by 80%, entirely because of a decrease in cell surface ENaC levels. Catalytically inactive mutants of PI5Kalpha 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 PI5KIalpha. Overexpression of epsin reduced amiloride-sensitive current in CCD cells. Overexpression of PI5KIalpha enhanced membrane PIP2 levels and reduced apical surface expression of ENaC in CCD cells, down-regulating amiloride-sensitive current. Knockdown of PI5KIalpha with isoform-specific siRNA resulted in a 4-fold enhancement of ENaC activity. PI5KIalpha 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 PI5KIalpha.
Weixel
Kelly M
KM
Department of Medicine, Renal and Electrolyte Division, University of Pittsburgh, Pittsburgh, Pennsylvania 15261, USA.
Edinger
Robert S
RS
Kester
Lauren
L
Guerriero
Christopher J
CJ
Wang
Huamin
H
Fang
Liang
L
Kleyman
Thomas R
TR
Welling
Paul A
PA
Weisz
Ora A
OA
Johnson
John P
JP
D033942:Adaptor Proteins, Vesicular Transport / Q000235:genetics / Q000378:metabolism; D000584:Amiloride / Q000494:pharmacology; D000818:Animals; D002461:Cell Line, Transformed; D002462:Cell Membrane / Q000201:enzymology; D002966:Clathrin / Q000235:genetics / Q000378:metabolism; D004705:Endocytosis / Q000187:drug effects / Q000502:physiology*; D053503:Epithelial Sodium Channels / Q000096:biosynthesis* / Q000235:genetics; D015870:Gene Expression; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D007685:Kidney Tubules, Collecting / Q000166:cytology / Q000502:physiology*; D008564:Membrane Potentials / Q000235:genetics / Q000502:physiology*; D051379:Mice; D015778:Minor Histocompatibility Antigens; D009865:Oocytes / Q000166:cytology; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000378:metabolism*; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D034741:RNA, Small Interfering; D026941:Sodium Channel Blockers / Q000494:pharmacology; D014982:Xenopus laevis
D033942:Adaptor Proteins, Vesicular Transport; D002966:Clathrin; D053503:Epithelial Sodium Channels; D007527:Isoenzymes; D015778:Minor Histocompatibility Antigens; D019269:Phosphatidylinositol 4,5-Diphosphate; D034741:RNA, Small Interfering; D026941:Sodium Channel Blockers; C114154:epsin; D000584:Amiloride; D017853:Phosphotransferases (Alcohol Group Acceptor); C402182:phosphatidylinositol phosphate 4-kinase