Nucleotide Specificity and Assessment of the Role of Phospholipids

We studied the ATP dependence of NHE-1, the ubiquitous isoform of the Na 1 /H 1 antiporter, using the whole-cell configuration of the patch-clamp technique to apply nucleotides intracellularly while measuring cytosolic pH (pH i ) by microfluorimetry. Na 1 /H 1 exchange activity was measured as the Na 1 -driven pH i 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 1 /H 1 exchange. Conversely, ATP perfusion restored exchange activity in cells that had been metabolically depleted by 2-deoxyD -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 z 5 mM Mg-ATP. Submillimolar concentrations failed to sustain Na 1 /H 1 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/Ca 2 1 exchanger, this requirement has been attributed to an aminophospholipid translocase, or “flippase.” The involvement of this enzyme in Na 1 /H 1 exchange was examined using fluorescent phosphatidylserine, which is actively translocated by the flippase. ATP depletion decreased the transmembrane uptake of NBD-labeled phosphatidylserine (NBDPS), 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 1 /H 1 exchange, implying that the effect of ATP is not mediated by changes in lipid distribution across the plasma membrane. K-ATP and ATP g 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 g S was ineffective. We conclude that ATP is the only soluble factor necessary for optimal activity of the NHE-1 isoform of the antiporter. Mg 2 1 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 g -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. key words: Na 1 /H 1 antiporter • phospholipid translocase • intracellular pH i n t r o d u c t i o n Na 1 /H 1 exchangers (NHEs), 1 or antiporters, are ubiquitous membrane transport proteins that play a major role in the regulation of intracellular pH (pH i ) and of cellular volume (Grinstein et al., 1985; Moolenaar, 1986; Demaurex and Grinstein, 1994). Under physiological conditions, NHEs catalyze the electroneutral exchange of extracellular Na 1 for intracellular H 1 , a process which is competitively inhibited by amiloride and its analogues (see Grinstein et al., 1989; Wakabayashi et al., 1992 b ). The rate of exchange is dictated largely by the state of protonation of an allosteric “modifier” site on the cytosolic side of the antiporter. Protonation of the modifier stimulates Na 1 /H 1 exchange, defending the cell from excessive acidification, while the occurrence of deprotonation near the physiological pH i deactivates the antiporter, precluding a potentially deleterious alkalinization of the cytosol (Aronson et al., 1982; Grinstein et al., 1984; Aronson, 1985). Na 1 /H 1 exchange activity has been observed in virtually all eukaryotic cells studied thus far. Yet, the kinetic and pharmacological properties of the exchange process vary widely between individual cell types and even between different domains of the plasma membrane in the case of asymmetric cells, e.g., the apical and basolateral membranes of epithelial cells (Haggerty et al., 1988). The structural basis of this functional diversity is attributed, at least in part, to the existence of distinct isoforms of the NHEs. In mammalian tissues, five separate isoforms have been identified to Address correspondence to Dr. Sergio Grinstein, Division of Cell Biology, Hospital for Sick Children, 555 University Avenue, Toronto, M5G 1X8, Canada. Fax: 416-813-5028; E-mail: [email protected] 1 Abbreviations used in this paper: CHO, Chinese hamster ovary; NBD, 7-nitrobenz-2-oxa-1,3-diazol-4-yl; NBD-PS, 1-C 6 -2-C 12 -NBD-phosphatidylserine; NHE, Na/H exchanger; pH i , intracellular pH; PS, phosphatidylserine. on Jne 5, 2017 D ow nladed fom Published February 1, 1997