title: Evidence that Na+/H+ exchanger 1 is an ATP-binding protein.
abstract: 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.
authors: Department of Molecular Physiology, National Cerebral and Cardiovascular Center Research Institute, Suita, Osaka, Japan.
authors: Naoko
authors: N
authors: Shimada-Shimizu
authors: Takashi
authors: T
authors: Hisamitsu
authors: Tomoe Y
authors: TY
authors: Nakamura
authors: Shigeo
authors: S
authors: Wakabayashi
mesh_terms: D000244:Adenosine Diphosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism*; D000818:Animals; D001386:Azides / Q000378:metabolism; D016367:Baculoviridae / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites; D002135:Calcium-Binding Proteins / Q000302:isolation & purification / Q000378:metabolism*; D027682:Cation Transport Proteins / Q000302:isolation & purification / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005822:Genetic Vectors / Q000235:genetics / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D008274:Magnesium / Q000378:metabolism; D019920:Photoaffinity Labels / Q000378:metabolism; D011485:Protein Binding; D025941:Protein Interaction Mapping; D059748:Proteolysis; D061987:Sf9 Cells; D012979:Sodium Radioisotopes / Q000378:metabolism; D000074082:Sodium-Hydrogen Exchanger 1; D017923:Sodium-Hydrogen Exchangers / Q000302:isolation & purification / Q000378:metabolism*; D014162:Transfection; D014466:Ultraviolet Rays
chemical_list: D001386:Azides; C469743:CHP1 protein, human; D002135:Calcium-Binding Proteins; D027682:Cation Transport Proteins; D019920:Photoaffinity Labels; C490998:SLC9A1 protein, human; D012979:Sodium Radioisotopes; D000074082:Sodium-Hydrogen Exchanger 1; D017923:Sodium-Hydrogen Exchangers; C017040:8-azidoadenosine 5'-triphosphate; D000244:Adenosine Diphosphate; D006160:Guanosine Triphosphate; D000255:Adenosine Triphosphate; D008274:Magnesium