The Interaction of Na+, K+, and Phosphate with the Gastric H,K-ATPase. Kinetics of E1-E2 Conformational Changes Assessed by Eosin Fluorescence Measurements.

H,K-ATPase and Na,K-ATPase show the highest degree of sequence similarity among all other members of the P-type ATPases family. To explore their common features in terms of ligand binding, we evaluated conformational transitions due to the binding of Na+, K+ and Pi in the H,K-ATPase, and compared the results with those obtained for the Na,K-ATPase. This work shows that eosin fluorescence time courses provide a reasonably precise method to study the kinetics of the E1-E2 conformational changes in the H,K-ATPase. We found that, although Na+ shifts the equilibrium toward the E1 conformation and seems to compete with H+ in ATPase activity assays, it was neither possible to isolate a Na+-occluded state, nor to reveal an influx of Na+ related to H,K-ATPase activity. The high rate of the E2K -> E1 transition found for the H,K-ATPase, which is not compatible with the presence of a K+-occluded form, agrees with the negligible level of occluded Rb+ (used as a K+ congener) found in the absence of added ligands. The use of vanadate and fluorinated metals to induce E2P-like states increased the level of occluded Rb+ and suggests that-during dephosphorylation-the probability of K+ to remain occluded increases from the E2P-ground to the E2P-product state. From kinetic experiments we found an unexpected increase in the values of k(obs) for E2P formation with [Pi]; consequently, to obey the Albers-Post model, the binding of Pi to the E2 state cannot be a rapid-equilibrium reaction.