ATP dependence of Na+/H+ exchange. Nucleotide specificity and assessment of the role of phospholipids.
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 approximately 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 gamma 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, GTP gamma S 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 gamma-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.
Demaurex
N
N
Division of Cell Biology, Hospital for Sick Children, Toronto, Montreal, Canada.
Romanek
R R
RR
Orlowski
J
J
Grinstein
S
S
D000255:Adenosine Triphosphate / Q000502:physiology*; D000818:Animals; D016466:CHO Cells; D002460:Cell Line; D006224:Cricetinae; D004247:DNA / Q000096:biosynthesis; D004594:Electrophysiology; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008564:Membrane Potentials / Q000502:physiology; D008856:Microscopy, Fluorescence; D009711:Nucleotides / Q000378:metabolism*; D018408:Patch-Clamp Techniques; D010743:Phospholipids / Q000378:metabolism*; D051381:Rats; D017923:Sodium-Hydrogen Exchangers / Q000378:metabolism*
D009711:Nucleotides; D010743:Phospholipids; D017923:Sodium-Hydrogen Exchangers; D000255:Adenosine Triphosphate; D004247:DNA