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

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.

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 Creators:
Geibel, Sven1, Author           
Kaplan, Jack H.2, Author
Bamberg, Ernst1, 3, Author           
Friedrich, Thomas1, 3, Author           
Affiliations:
1Department of Biophysical Chemistry, Max Planck Institute of Biophysics, Max Planck Society, ou_2068289              
2Oregon Health Science University, 3181 Southwest Sam Jackson Park Road, Portland, OR 97201, ou_persistent22              
3Chemical and Pharmaceutical Sciences Department, Johann Wolfgang Goethe University Frankfurt, Frankfurt, Germany, ou_persistent22              

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 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.

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Language(s): eng - English
 Dates: 2002-07-182002-12-032003-01-272003-02-04
 Publication Status: Published in print
 Pages: 6
 Publishing info: -
 Table of Contents: -
 Rev. Type: Peer
 Identifiers: DOI: 10.1073/pnas.0337336100
 Degree: -

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Title: Proceedings of the National Academy of Sciences of the United States of America
  Other : PNAS
  Other : Proceedings of the National Academy of Sciences of the USA
  Abbreviation : Proc. Natl. Acad. Sci. U. S. A.
Source Genre: Journal
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Publ. Info: Washington, D.C. : National Academy of Sciences
Pages: - Volume / Issue: 100 (3) Sequence Number: - Start / End Page: 964 - 969 Identifier: ISSN: 0027-8424
CoNE: https://pure.mpg.de/cone/journals/resource/954925427230