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Conformational dynamics of the Na+/K+-ATPase probed by voltage clamp fluorometry

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Geibel,  Sven
Department of Biophysical Chemistry, Max Planck Institute of Biophysics, Max Planck Society;

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Bamberg,  Ernst
Department of Biophysical Chemistry, Max Planck Institute of Biophysics, Max Planck Society;
Chemical and Pharmaceutical Sciences Department, Johann Wolfgang Goethe University Frankfurt, Frankfurt, Germany;

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Friedrich,  Thomas
Department of Biophysical Chemistry, Max Planck Institute of Biophysics, Max Planck Society;
Chemical and Pharmaceutical Sciences Department, Johann Wolfgang Goethe University Frankfurt, Frankfurt, Germany;

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Citation

Geibel, S., Kaplan, J. H., Bamberg, E., & Friedrich, T. (2003). Conformational dynamics of the Na+/K+-ATPase probed by voltage clamp fluorometry. Proceedings of the National Academy of Sciences of the United States of America, 100(3), 964-969. doi:10.1073/pnas.0337336100.


Cite as: https://hdl.handle.net/11858/00-001M-0000-0024-DBD0-D
Abstract
The method of voltage clamp fluorometry combined with site-directed fluorescence labeling was used to detect local protein motions of the fully active Na+/K+-ATPase in real time under physiological conditions. Because helix M5 extends from the cytoplasmic site of ATP hydrolysis into the cation binding region, we chose the extracellular M5–M6 loop of the sheep α1-subunit for the insertion of cysteine residues to identify reporter positions for conformational rearrangements during the catalytic cycle. After expression of the single cysteine mutants in Xenopus oocytes and covalent attachment of tetramethylrhodamine-6-maleimide, only mutant N790C reported molecular rearrangements of the M5–M6 loop by showing large, ouabain-sensitive fluorescence changes (≈5%) on addition of extracellular K+. When the enzyme was subjected to voltage jumps under Na+/Na+-exchange conditions, we observed fluorescence changes that directly correlated to transient charge movements originating from the E1P–E2P transition of the transport cycle. The voltage jump-induced fluorescence changes and transient currents were abolished after replacement of Na+ by tetraethylammonium or on addition of ouabain, showing that conformational flexibility is impaired under these conditions. Voltage-dependent fluorescence changes could also be observed in the presence of subsaturating K+ concentrations. This allowed to monitor the time course of voltage-dependent relaxations into a new stationary distribution of states under turnover conditions, showing the acceleration of relaxation kinetics with increasing K+ concentrations. As a result, the stationary distribution between E1 and E2 states and voltage-dependent relaxation times can be determined at any time and membrane potential under Na+/Na+ exchange as well as Na+/K+ turnover conditions.