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

Na+/H+ exchange is a passive process not requiring expenditure of metabolic energy. Nevertheless, depletion of cellular ATP produces a marked inhibiti on of the antiport. No evidence has been found for direct binding of nucleo tide to exchangers or alteration in their state of phosphorylation, suggest ing ancillary factors may be involved. This possibility was tested by compa ring the activity of dog red blood cells (RBC) and their resealed ghosts. I mmunoblotting 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 b y submicromolar concentrations of the benzoyl guanidinium compound HOE-694, consistent with mediation by NHE1. Although exchange activity could also b e 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 contin ued export of base equivalents by the endogenous anion exchanger. These obs ervations suggest a critical factor required to maintain optimal Na+/H+ exc hange activity is lost or inactivated during preparation of ghosts. Depleti on of ATP, achieved by incubation with 2-deoxy-D-glucose, inhibited Na+/Hexchange in intact RBC, as reported for nucleated cells. In contrast, the r ate of exchange was similar in control and ATP-depleted resealed ghosts. In terestingly, the residual rate of Na+/H+ exchange in ATP-depleted but other wise intact cells was similar to the transport rate of ghosts. Therefore, w e tentatively conclude that full activation of NHE1 requires both ATP and a n additional regulatory factor, which may mediate the action of the nucleot ide. Ancillary phosphoproteins or phospholipids or the kinases that mediate their phosphorylation are likely candidates for the regulatory factor(s) t hat is inactivated or missing in ghosts.