Endosomal Recycling of the Na+/H+Exchanger NHE3 Isoform Is Regulated by the Phosphatidylinositol 3-Kinase Pathway*
- From the ‡Cell Biology Programme, Research Institute, Hospital for Sick Children, Toronto, Ontario M5G 1X8, Canada and the§Department of Physiology, McGill University, Montréal, Québec H3G 1Y6, Canada
Abstract
The NHE3 isoform of the Na+/H+ exchanger localizes to both the plasmalemmal and endosomal compartments in polarized epithelial and transfected Chinese hamster ovary (AP-1) cells. It is unclear how the distribution of NHE3 between these compartments is regulated. In this study, we examined the potential involvement of phosphatidylinositol 3′-kinase (PI3-K) in regulating the activity and distribution of NHE3, as this lipid kinase has been implicated in modulating vesicular traffic in the endosomal recycling pathway. Wortmannin and LY294002, both potent inhibitors of PI3-K, markedly inhibited NHE3-mediated H+ extrusion across the plasma membrane in a concentration- and time-dependent manner. The subcellular distribution of the antiporters was monitored by transfecting epitope-tagged NHE3 into AP-1 cells. In parallel with the inhibition of transport, PI3-K antagonists induced a pronounced loss of NHE3 from the cell surface and its accumulation in an intracellular compartment, as assessed by immunofluorescence microscopy and enzyme-linked immunosorbent assays. Further analysis using cells transfected with antiporters bearing an external epitope tag revealed that the redistribution reflected primarily a decrease in the rate of recycling of intracellular NHE3 to the cell surface. The wortmannin-induced inhibition and redistribution of NHE3 were prevented when cells were incubated at 4 °C, consistent with the known temperature dependence of the endocytic process. These observations demonstrate that NHE3 activity is controlled by dynamic endocytic and recycling events that are modulated by PI3-K.
Footnotes
-
↵* This work was supported in part by the Medical Research Council of Canada and the Kidney Foundation of Canada.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
-
↵¶ Supported by a research scholarship from the Fonds de la Recherche en Santé du Quebec.
-
↵‖ International Scholar of the Howard Hughes Medical Institute and current holder of the Pitblado Chair in Cell Biology. Cross-appointed to the Department of Biochemistry of the University of Toronto. To whom correspondence should be addressed: Div. of Cell Biology, Hospital for Sick Children, 555 University Ave., Toronto M5G 1X8, Canada. Tel.: 416-813-5727; Fax: 416-813-5028; E-mail:sga{at}sickkids.on.ca.
-
↵2 It is conceivable that some of the internalized NHE3 recycled back to the surface during the period studied. In this event, inhibition of exocytosis would appear as enhanced endocytosis.
-
↵3 K. Kurashima, unpublished observations.
- Abbreviations:
- NHE
-
Na+/H+ exchanger
- BCECF
-
2′,7′-bis(2-carboxyethyl)-5(6)-carboxyfluorescein
- CHO
-
Chinese hamster ovary
- ELISA
-
enzyme-linked immunosorbent assay
- HA
-
hemagglutinin
- αMEM
-
α-minimal essential medium
- PBS
-
phosphate-buffered saline
- pHi
-
intracellular (cytosolic) pH
- PI3-K
-
phosphatidylinositol 3’-kinase
- PAGE
-
polyacrylamide gel electrophoresis.
-
- Received May 13, 1998.
- Revision received June 4, 1998.
- The American Society for Biochemistry and Molecular Biology, Inc.
