Both Na,K-ATPase and Ca-ATPase are critical cation transport systems in membranes of eucaryotic cells. The plasma membrane Na,K-ATPase pumps Na+ out of cells in exchange for K+. Sarcoplasmic reticulum Ca-ATPase is important for lowering intracellular Ca2+ in the heart between contractions. Increasing our insight into the mechanism and the regulation of these enzymes is of widespread interest at the molecular, the membrane, the cellular, and the whole animal level. The most fundamental questions involve 1) the free energy coupling between ATP hydrolysis and ion transport, 2) the molecular structure of the ion channels, and 3) the regulation of enzyme activity to meet the needs of the cell and the organism. Insight into these mechanisms may be crucial for our understanding of disease and aging. In a series of experiments, we have gained insight into the nature and mechanism of the three Na+ binding sites within the ion channel of the Na,K-ATPase. A model for Na+ transport by electric organ Na,K-ATPase is proposed based on the statistical partitioning of Na+ binding states. The model accounts for (1) the biphasic time course of phosphorylation obtained under conditions where Na+ is added last, and (2) the conversion to monophasic kinetics when the enzyme is preincubated with Na+ prior to ATP addition. These results imply that the third Na+ binding site is restricted when the other two sites are occupied. In other experiments we examined the coupling of Ca transport to ATP hydrolysis in isolated cardiac sarcoplasmic reticulum. Quenched-flow measurements of phosphoenzyme formation by the Ca2+ pump in sarcoplasmic reticulum (SR) were performed at 2xC for comparison with EPR (electron spin resonance) spectral data demonstrating a slow conformational change associated with active Ca2+ transport. Phosphoenzyme formation was biphasic with a close correlation between the slow phase of phosphorylation and the EPR spectral change. The results indicate that the spin label senses a protein conformational change linked to phosphorylation of the Ca2+ ATPase or to the interconversion of subsequently-generated phosphorylated intermediate states. These studies provide important information about the mechanism for free energy coupling between ATP hydrolysis and Ca2+ transport.
