1-ANILINO-8-NAPHTALENE SULFONATE PROBES A GASTRIC HK-ATPASE POTASSIUMSITE WHOSE ACCESS REQUIRES IONOPHORES

1-anilino-8-naphtalenesulfonate (ANS) is a hydrophobic dipole previous ly used to demonstrate that the proton for potassium exchange by the g astric HK-ATPase is electroneutral. In this paper, we demonstrate that ANS binds to gastric membranes and probes conformational changes of t he HK-ATPase independently of any active H for K exchange. Conformatio nal changes require the presence of potassium-valinomycin and are not triggered by sodium. Potassium effect is enhanced by ATP, in the prese nce and in the absence of magnesium and, by ADP, in the presence of ma gnesium. Labeling of the pig HK-ATPase K518 by fluorescein-5-isothiocy anate inhibits the enzyme activity and knocks out the ATP effect on AN S fluorescence. Scherring 28080 and the monoclonal antibody 95-111, tw o competitive inhibitors of K-activated ATPase dephosphorylation, do n ot modify K-effect on ANS fluorescence but inhibit ATP effects. This s upports that ANS does not probe K-site between the H1-H2 loop. Treatme nt of gastric membranes with trypsin does not inhibit the ANS response to potassium but does inhibit the response to ATP. This suggests that the ATP site inducing the ANS response is cytoplasmic and the potassi um site is intramembranous. Titration reveals that one mole of ANS int eracts with one mole of ATPase. We suggest that ANS probes a hydrophob ic potassium site of gastric ATPase and that addition of ATP and ADP-M g embed that site.