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Published online 10 July 2000. doi:10.1083/jcb.150.1.213
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© The Rockefeller University Press, 0021-9525/2000//213 $5.00
The Journal of Cell Biology, Volume 150, Number 1, , 2000 213-224

Original Article

Intracellular Ph Regulation by Na+/H+ Exchange Requires Phosphatidylinositol 4,5-Bisphosphate



Orit Aharonovitza, Hans C. Zaunb, Tamas Ballac, John D. Yorkd, John Orlowskib, and Sergio Grinsteina

a Cell Biology Programme, Research Institute, The Hospital for Sick Children, Toronto, Ontario M5G 1X8, Canada
b Department of Physiology, McGill University, Montréal, Québec, H3G 1Y6, Canada
c Endocrinology and Reproduction Research Branch, National Institutes of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892-4510
d Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, North Carolina 27710
Division of Cell Biology, Hospital for Sick Children, 555 University Avenue, Toronto, M5G 1X8, Canada.(416) 813-5028(416) 813-5727

sga{at}sickkids.on.ca

The carrier-mediated, electroneutral exchange of Na+ for H+ across the plasma membrane does not directly consume metabolic energy. Nevertheless, acute depletion of cellular ATP markedly decreases transport. We analyzed the possible involvement of polyphosphoinositides in the metabolic regulation of NHE1, the ubiquitous isoform of the Na+/H+ exchanger. Depletion of ATP was accompanied by a marked reduction of plasmalemmal phosphatidylinositol 4,5-bisphosphate (PIP2) content. Moreover, sequestration or hydrolysis of plasmalemmal PIP2, in the absence of ATP depletion, was associated with profound inhibition of NHE1 activity. Examination of the primary structure of the COOH-terminal domain of NHE1 revealed two potential PIP2-binding motifs. Fusion proteins encoding these motifs bound PIP2 in vitro. When transfected into antiport-deficient cells, mutant forms of NHE1 lacking the putative PIP2-binding domains had greatly reduced transport capability, implying that association with PIP2 is required for optimal activity. These findings suggest that NHE1 activity is modulated by phosphoinositides and that the inhibitory effect of ATP depletion may be attributable, at least in part, to the accompanying net dephosphorylation of PIP2.

Key Words: amiloride • ATP depletion • Na+/H+ antiport • phosphoinositide

© 2000 The Rockefeller University Press

Abbreviations used in this paper: AM, acetoxymethylester; BCECF, 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein; Ca2+i, cytosolic free calcium; ERM, ezrin/radixin/moesin; GFP, green fluorescent protein; GST, glutathione-S-transferase; IP3, 1,4,5,-trisphosphate; HA, hemagglutinin; NHE, Na+/H+ exchanger; NMG, N-methyl-D-glucammonium; pHi, intracellular pH; PH, pleckstrin homology; PIP2, phosphatidylinositol-4,5-bisphosphate; PLC, phospholipase C; TPCK, N-tosyl-l-phenylalanine chloromethyl ketone.


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