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The plasma membrane Na(+)/H(+) exchanger 1 (NHE1) is rapidly activated in response to various stimuli. The membrane-proximal cytoplasmic region (∼60 residues), termed the lipid-interacting domain (LID), is an important regulatory domain of NHE1. Here, we used a pharmacological approach to further characterize the role of LID in the regulation of NHE1. Pharmacological analysis using staurosporine-like indolocarbazole and bisindolylmaleimide compounds suggested that the phorbol ester- and receptor agonist-induced activation of NHE1 occurs through a protein kinase C-independent mechanism. In particular, only indolocarbazole compounds that inhibited NHE1 activation were able to interact with the LID, suggesting that the inhibition of NHE1 activation is achieved through the direct action of these compounds on the LID. Furthermore, in addition to phorbol esters and a receptor agonist, okadaic acid and hyperosmotic stress, which are known to activate NHE1 through unknown mechanisms, were found to promote membrane association of the LID concomitant with NHE1 activation; these effects were inhibited by staurosporine, as well as by a mutation in the LID. Binding experiments using the fluorescent ATP analog trinitrophenyl ATP revealed that ATP and the NHE1 activator phosphatidylinositol 4,5-bisphosphate bind competitively to the LID. These findings suggest that modulation of NHE1 activity by various activators and inhibitors occurs through the direct binding of these molecules to the LID, which alters the association of the LID with the plasma membrane.

Na(+)/H(+) exchanger (NHE) 1 is a member of the solute carrier superfamily, which regulates intracellular ionic homeostasis. NHE1 is known to require cellular ATP for its activity, despite there being no requirement for energy input from ATP hydrolysis. In this study, we investigated whether NHE1 is an ATP-binding protein. We designed a baculovirus vector carrying both epitope-tagged NHE1 and its cytosolic subunit CHP1, and expressed the functional NHE1-CHP1 complex on the surface of Sf9 insect cells. Using the purified complex protein consisting of NHE1 and CHP1 from Sf9 cells, we examined a photoaffinity labeling reaction with 8-azido-ATP-biotin. UV irradiation promoted the incorporation of 8-azido-ATP into NHE1, but not into CHP1, with an apparent Kd of 29.1 µM in the presence of Mg(2+). The nonlabeled nucleotides ATP, GTP, TTP and CTP all inhibited this crosslinking. However, ATP had the strongest inhibitory effect, with an apparent inhibition constant (IC50) for ATP of 2.2 mM, close to the ATP concentration giving the half-maximal activation of NHE1 activity. Importantly, crosslinking was more strongly inhibited by ATP than by ADP, suggesting that ATP is dissociated from NHE1 upon ATP hydrolysis. Limited proteolysis with thrombin and deletion mutant analysis revealed that the 8-azido-ATP-binding site is within the C-terminal cytoplasmic domain of NHE1. Equilibrium dialysis with NHE1-derived peptides provided evidence that ATP directly binds to the proximal cytoplasmic region (Gly542-Pro598), which is critical for ATP-dependent regulation of NHE1. These findings suggest that NHE1 is an ATP-binding transporter. Thus, ATP may serve as a direct activator of NHE1.