Open Access

Stefan Geys

• The Na+,K+-ATPase was discovered more than 50 years ago, but even today the pumpcycle and its partial reactions are still not completely understood. In this thesis, Voltage Clamp Fluorometry was used to monitor the conformational changes that are associated with several electrogenic partial reactions of the Na+,K+-ATPase. The conformational dynamics of the ion pump were analyzed at different concentrations of internal Na+ or of external K+ and the influences on the conformational equilibrium were determined. To probe the effect of the internal Na+ concentration on the Na+ branch of the ion pump, oocytes were first depleted of internal Na+ and then loaded with Na+ using the epithelial sodium channel which can be blocked by amiloride. The conformational dynamics of the K+ branch were studied using different external K+ concentrations in the presence and in the absence of external Na+ to yield additional information on the apparent affinity of K+. The results of our Voltage Clamp Fluorometry experiments demonstrate that lowering the intracellular concentration of Na+ has a comparable effect on the conformational equilibrium as increasing the amount of K+ in the external solution. Both of these changes shift the equilibrium towards the E1/E1(P) conformation. Furthermore, it can be shown that the ratio between external Na+ and K+ ions is also a determinant for the position of the conformational equilibrium: in the absence of external Na+, the K+ dependent shift of the equilibrium towards E1 was observed at a much lower K+ concentration than in the presence of Na+. In addition, indications were found that both external K+ and internal Na+ bind within an ion well. Finally, the crucial role of negatively charged glutamate residues in the 2nd extracellular loop for the control of ion-access to the binding sites could be verified.
• In der vorliegenden Arbeit wurden die Methoden der ortsspezifischen Fluoreszenzmarkierung und der Voltage Clamp Fluorometrie (VCF) kombiniert um die Konformationsänderungen der Na+,K+-ATPase während verschiedener elektrogener Reaktionsschritte des Pumpzyklus zu untersuchen. Neben der extrazellulären Freisetzung von Na+ waren vor allem die interne Na+-Bindung und externe K+-Bindung von Interesse, da diese aufgrund ihrer geringen Elektrogenizität mit rein elektrophysiologischen Methoden nicht, oder nur ungenügend, erfasst werden können. Es sollten vor allem Informationen über die Dynamik der mit den Reaktionsschritten assoziierten Konformationsänderungen gewonnen werden und der Einfluss der Pumpsubstrate Na+ und K+ auf die Lage des Konformationsgleichgewichts geklärt werden. ...