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

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

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Zimmermann,  Dirk
Department of Biophysical Chemistry, Max Planck Institute of Biophysics, Max Planck Society;

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Zifarelli,  Giovanni
Department of Biophysical Chemistry, Max Planck Institute of Biophysics, Max Planck Society;

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Becker,  Anja
Department of Biophysical Chemistry, Max Planck Institute of Biophysics, Max Planck Society;

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Friedrich,  Thomas
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;

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Citation

Geibel, S., Zimmermann, D., Zifarelli, G., Becker, A., Koenderink, J. B., Hu, Y.-K., et al. (2003). Conformational dynamics of Na+/K+- and H+/K+-ATPase probed by voltage clamp fluorometry. Annals of the New York Academy of Sciences, 986(1), 31-38. doi:10.1111/j.1749-6632.2003.tb07136.x.


Cite as: http://hdl.handle.net/11858/00-001M-0000-0024-DBD2-9
Abstract
We used the method of site-directed fluorescence labeling in combination with voltage-clamp fluorometry for time-resolved recording of localized conformational transitions of the Na+/K+- and H+/K+-ATPase. Therefore, single cysteine mutations were introduced into the extracellular TM5-TM6 loop of the sheep Na+/K+-ATPase alpha1-subunit devoid of other extracellular cysteines. Upon expression in Xenopus oocytes and covalent attachment of tetramethylrhodamine-maleimide (TMRM) as a reporter fluorophore, Cys-mutant N790C showed large fluorescence changes of up to 5% in response to extracellular K+ that were completely abolished by ouabain. When voltage jumps were applied under Na+/Na+-exchange conditions, we observed fluorescence changes that paralleled the transient currents originating from the E1P<-->E2P transition. These fluorescence changes were also completely inhibited by ouabain, as were the voltage jump-induced transient currents. Transient fluorescence changes could also be measured as a function of increasing K+ concentrations, that is, under turnover conditions. As a result, the distribution between E1 and E2 states can be determined at any time and membrane potential. Very similar fluorescence signals were obtained for rat gastric H+/K+-ATPase upon expression in oocytes, when a single cysteine was introduced at a position homologous to N790 in Na+/K+-ATPase for attachment of the fluorophore. As to the high sequence similarity among P-type ATPases within the TM5 helix and the TM5-TM6 loop region, our results enable new means of kinetic investigation for these pumps under physiological conditions in living cells.