Voltage Dependent Conformational Changes of the Na+/K+-ATPase Revealed by Site Directed Fluorometry

Abstract

Most of the voltage dependence of the Na+/K+-ATPase cycle originates from steps associated with extracellular Na+ binding/release and occlusion/deocclusion transitions. In order to explore regions of the Na+/K+ pump that respond to voltage, we engineered single cysteine mutants facing the outside of the α and β subunits as targets of cysteine-reactive tetramethyl rhodamine (TMRM). These pumps were expressed in Xenopus oocytes and voltage-clamped to obtain simultaneous electrical and optical recordings. We have detected voltage dependent fluorescence changes when TMRM is conjugated at sites of the α and β subunits. Interestingly, positions within the β subunit produced robust voltage dependent fluorescence signals. These fluorescence changes follow the kinetics of Na+ translocation through the pump. Quenching of the fluorescence signal can be altered by tryptophan mutations at the external side of the α subunit, indicating movement of the external face of the α subunit. Surprisingly, in some positions fluorescence changes persist even in the presence of ouabain, a specific inhibitor of the pump, and these changes are modified by tyrosine mutations in the transmembrane segment of the β subunit. These results suggest that there are intrinsic voltage-dependent conformational changes in the Na+/K+ pump. Supported by U54GM087519 and GM030376.