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+ exchang er (NHE), yet depletion of the nucleotide drastically reduces the rate of cation exchange. The mechanism underlying this inhibition was inve stigated in fibroblasts transfected with NHE-1, the growth factor-sens itive isoform of the antiport. NHE-1 was found to be phosphorylated in serum-starved, unstimulated cells. Acute ATP depletion induced a prof ound inhibition of transport without detectable changes in NHE-1 phosp horylation. Analysis of cells transfected with truncated mutants of NH E-1 indicated that the carboxyl-terminal cytosolic domain of the antip ort is required for expression of its ATP dependence. To define whethe r 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 compa rable before and after ATP depletion. Immunofluorescence determination s revealed focal accumulations of NHE-1 on the membrane of untreated c ells. NHE-1 redistributed following ATP depletion, showing a more homo geneous localization. F-actin, which co-localizes with the antiport in untreated cells, also redistributed when cells were ATP depleted. The se findings suggest an interaction of NHE-1 with the cytoskeleton. Acc ordingly, disassembly of actin filaments with cytochalasin D induced r edistribution of the antiport. However, Na+/H+ exchange activity was u naltered 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 in teraction between NHE-1 and the putative regulator, inhibiting Na+/Hexchange and inducing subcellular redistribution. However, disruption of the cytoskeleton at distal sites, such as induced by cytochalasins, is insufficient to inactivate the antiport.