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From the * Division of Cell Biology, Hospital for Sick Children, Toronto, M5G 1X8; and We studied the ATP dependence of NHE-1, the ubiquitous isoform of the Na+/H+ antiporter, using
the whole-cell configuration of the patch-clamp technique to apply nucleotides intracellularly while measuring cytosolic pH (pHi) by microfluorimetry. Na+/H+ exchange activity was measured as the Na+-driven pHi recovery
from an acid load, which was imposed via the patch pipette. In Chinese hamster ovary (CHO) fibroblasts stably
transfected with NHE-1, omission of ATP from the pipette solution inhibited Na+/H+ exchange. Conversely, ATP
perfusion restored exchange activity in cells that had been metabolically depleted by 2-deoxy-D-glucose and oligomycin. In cells dialyzed in the presence of ATP, no "run-down" was observed even after extended periods, suggesting that the nucleotide is the only diffusible factor required for optimal NHE-1 activity. Half-maximal activation of
the antiporter was obtained at ~5 mM Mg-ATP. Submillimolar concentrations failed to sustain Na+/H+ exchange
even when an ATP regenerating system was included in the pipette solution. High ATP concentrations are also
known to be required for the optimal function of other cation exchangers. In the case of the Na/Ca2+ exchanger,
this requirement has been attributed to an aminophospholipid translocase, or "flippase." The involvement of this
enzyme in Na+/H+ exchange was examined using fluorescent phosphatidylserine, which is actively translocated
by the flippase. ATP depletion decreased the transmembrane uptake of NBD-labeled phosphatidylserine (NBD-PS), indicating that the flippase was inhibited. Diamide, an agent reported to block the flippase, was as potent as
ATP depletion in reducing NBD-PS uptake. However, diamide had no effect on Na+/H+ exchange, implying that
the effect of ATP is not mediated by changes in lipid distribution across the plasma membrane. K-ATP and ATP Department of Physiology, McGill University,
Montreal, H3G 1Y6, Canada
S
were as efficient as Mg-ATP in sustaining NHE-1 activity, while AMP-PNP and AMP-PCP only partially substituted
for ATP. In contrast, GTPS was ineffective. We conclude that ATP is the only soluble factor necessary for optimal
activity of the NHE-1 isoform of the antiporter. Mg2+ does not appear to be essential for the stimulatory effect of ATP. We propose that two mechanisms mediate the activation of the antiporter by ATP: one requires hydrolysis
and is likely an energy-dependent event. The second process does not involve hydrolysis of the -phosphate, excluding mediation by protein or lipid kinases. We suggest that this effect is due to binding of ATP to an as yet unidentified, nondiffusible effector that activates the antiporter.
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