ATP dependence of NHE-1, the ubiquitous isoform of the Na+/H+ antiporter. Analysis of phosphorylation and subcellular localization.

ATP is not hydrolyzed during the transport cycle of the Na+/H+ exchanger (NHE), yet depletion of the nucleotide drastically reduces the rate of cation exchange. The mechanism underlying this inhibition was investigated in fibroblasts transfected with NHE-1, the growth factor-sensitive isoform of the antiport. NHE-1 was found to be phosphorylated in serum-starved, unstimulated cells. Acute ATP depletion induced a profound inhibition of transport without detectable changes in NHE-1 phosphorylation. Analysis of cells transfected with truncated mutants of NHE-1 indicated that the carboxyl-terminal cytosolic domain of the antiport is required for expression of its ATP dependence. To define whether inhibition of Na+/H+ exchange resulted from internalization of NHE-1, extracellularly exposed proteins were labeled with impermeant biotin derivatives. The proportion of NHE-1 exposed to the surface was comparable before and after ATP depletion. Immunofluorescence determinations revealed focal accumulations of NHE-1 on the membrane of untreated cells. NHE-1 redistributed following ATP depletion, showing a more homogeneous localization. F-actin, which co-localizes with the antiport in untreated cells, also redistributed when cells were ATP depleted. These findings suggest an interaction of NHE-1 with the cytoskeleton. Accordingly, disassembly of actin filaments with cytochalasin D induced redistribution of the antiport. However, Na+/H+ exchange activity was unaltered by cytochalasin D. We propose that ancillary proteins confer ATP sensitivity to the antiporter and may also mediate its association with the cytoskeleton. Depletion of the nucleotide would alter the interaction between NHE-1 and the putative regulator, inhibiting Na+/H+ exchange and inducing subcellular redistribution. However, disruption of the cytoskeleton at distal sites, such as induced by cytochalasins, is insufficient to inactivate the antiport.