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Am J Physiol Cell Physiol 276: C1303-C1311, 1999;
0363-6143/99 $5.00
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Vol. 276, Issue 6, C1303-C1311, June 1999

ATP dependence is not an intrinsic property of Na+/H+ exchanger NHE1: requirement for an ancillary factor

Orit Aharonovitz, Nicolas Demaurex, Michael Woodside, and Sergio Grinstein

Division of Cell Biology, Hospital for Sick Children, Toronto, Ontario, Canada M5G 1X8

Na+/H+ exchange is a passive process not requiring expenditure of metabolic energy. Nevertheless, depletion of cellular ATP produces a marked inhibition of the antiport. No evidence has been found for direct binding of nucleotide to exchangers or alteration in their state of phosphorylation, suggesting ancillary factors may be involved. This possibility was tested by comparing the activity of dog red blood cells (RBC) and their resealed ghosts. Immunoblotting experiments using isoform-specific polyclonal and monoclonal antibodies indicated RBC membranes express Na+/H+ exchanger isoform 1 (NHE1). In intact RBC, uptake of Na+ was greatly stimulated when the cytosol was acidified. The stimulated uptake was largely eliminated by amiloride and by submicromolar concentrations of the benzoyl guanidinium compound HOE-694, consistent with mediation by NHE1. Although exchange activity could also be elicited by acidification in resealed ghosts containing ATP, the absolute rate of transport was markedly diminished at comparable pH. Dissipation of the pH gradient was ruled out as the cause of diminished transport rate in ghosts. This was accomplished by a "pH clamping" procedure based on continued export of base equivalents by the endogenous anion exchanger. These observations suggest a critical factor required to maintain optimal Na+/H+ exchange activity is lost or inactivated during preparation of ghosts. Depletion of ATP, achieved by incubation with 2-deoxy-D-glucose, inhibited Na+/H+ exchange in intact RBC, as reported for nucleated cells. In contrast, the rate of exchange was similar in control and ATP-depleted resealed ghosts. Interestingly, the residual rate of Na+/H+ exchange in ATP-depleted but otherwise intact cells was similar to the transport rate of ghosts. Therefore, we tentatively conclude that full activation of NHE1 requires both ATP and an additional regulatory factor, which may mediate the action of the nucleotide. Ancillary phosphoproteins or phospholipids or the kinases that mediate their phosphorylation are likely candidates for the regulatory factor(s) that is inactivated or missing in ghosts.

pH regulation; amiloride; red blood cells; ghosts


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