Single-Molecule Measurements to Investigate the Negative Cooperativity in Na+/K+ -ATPase

Abstract

The Na+/K+-ATPase, a cell membrane ion motive ATPase, uses energy from the hydrolysis of ATP to move Na+ out of and K+ into cells, thus maintaining the membrane resting potential and cellular volume. To investigate how this pump functions, we isolated ATPase from duck supraorbital salt glands and labeled it with Cy3-maleimide (Cy3-ATPase). In bulk experiments, we found that the fluorescence of Cy3-ATPase decreases in the presence of ATP (Biochim Biophys Acta 2009; 1794:1549-1557). The kinetics of this ATP-induced fluorescence decrease exhibited negative cooperativity and could be explained in terms of protein aggregation. To further explore the phenomenon of negative cooperativity on the level of individual monomers, we used single-molecule total internal reflection fluorescence (SM-TIRF) microscopy. Protein monomers were solubilized and reconstituted into lipid vesicles to investigate the effect of varying ATP concentration on the fluorescence.Data from SM-TIRF experiments, analyzed using a hidden Markov model (HMM), suggest that the Cy3-ATPase exists in dynamic equilibrium between a high fluorescence state (unquenched) and a low fluorescence state (partially quenched). These kinetics are characterized by either rapid or slow transitions between these states. Two subpopulations are observed, one where the transitions between the states occur rapidly and the other where the kinetics are slower. Preliminary analysis of the data suggests that ATP shifts the population distribution from those exhibiting rapid transitions to those exhibiting slow transitions. Here, we report on the analysis of these effects and the implications of the above observations on the working of the pump.