Recently we have presented evidence that the fluorescence probe RH 421 can be used to detect binding and release of ions at the extracellular face of the pump since these processes are associated with translocation of electrical charge [1]. Applying this method to experiments with cardiac glycosides we found that: (1) ouabain induced fluorescence changes of the electrochromic dye, RH 421, were caused by the changes of charges bound to the enzyme: (2) independent of the sodium concentration, the final fluorescence amplitude indicated that approximately 2 Na+ ions were bound to the pump: (3) the sodium release to the extracellular side involved two distinct electrogenic steps: (4) the kinetics of inhibition depended on the Na+ concentration. Experiments with hydrophobic ions indicated that the kinetics of ouabain binding to the Na-ATPase is voltage dependent and (5) the applied techniques is a convenient tool to characterized binding of cardiac glycosides to the Na,K-pump.
