+ 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 K d 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 (IC 50 ) 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.

The plasma membrane Na 1 /H 1 exchanger 1 (NHE1) is rapidly activated in response to various stimuli. The membraneproximal cytoplasmic region (∼60 residues), termed the lipidinteracting 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. IntroductionThe ubiquitous Na 1 /H 1 exchanger isoform 1 (NHE1) catalyzes acid extrusion across the cell membrane by electroneutral ion exchange coupled to Na 1 influx and serves as a key regulator of intracellular pH (pH i ), Na 1 concentration, and cell volume in virtually all tissues (Wakabayashi et al., 1997;Orlowski and Grinstein, 2004). NHE1 elicits relatively high exchange activity under unstimulated physiologic conditions (basal state) and is further activated in response to various stimuli, including hormones, growth factors, and mechanical stress (activated state). This activation is attributable to a change in its affinity for intracellular H 1 ; thus, it can be easily detected in the neutral pH i range as a stimuli-induced cytoplasmic alkalinization in the absence of bicarbonate (Wakabayashi et al., 1997). Although NHE1 activation plays a physiologically important role in optimizing the intracellular ionic environment, it is also thought to promote the pathogenesis of disease, such as heart failure and cancer (Cardone et al., 2005;Karmazyn et al., 2008;Wakabayashi et al., 2013). In fact, treatment with NHE1 inhibitors reduced the pathologic phenotypes of animal models with acute and chronic heart diseases (Karmazyn et al., 2008).The molecular mechanism of the hormonal activation of NHE1 has been the focus of many investigations. The stimulation of Gq-coupled receptors induces the hydrolysis of phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P 2 ] via the a...