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  Electrophysiological analysis of the mutated Na,K-ATPase cation binding pocket

Koenderink, J. B., Geibel, S., Grabsch, E., De Pont, J. J. H., Bamberg, E., & Friedrich, T. (2003). Electrophysiological analysis of the mutated Na,K-ATPase cation binding pocket. The Journal of Biological Chemistry, 278(51), 51213-51222. doi:10.1074/jbc.M306384200.

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 Creators:
Koenderink, Jan B.1, Author           
Geibel, Sven1, Author           
Grabsch, Eva1, Author           
De Pont, Jan Joep H.H.M.2, Author
Bamberg, Ernst1, Author           
Friedrich, Thomas1, Author           
Affiliations:
1Department of Biophysical Chemistry, Max Planck Institute of Biophysics, Max Planck Society, ou_2068289              
2Department of Biochemistry, University Medical Center Nijmegen, P. O. Box 9101, 6500 HB Nijmegen, The Netherlands, ou_persistent22              

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 Abstract: Na,K-ATPase mediates net electrogenic transport by extruding three Na+ ions and importing two K+ ions across the plasma membrane during each reaction cycle. We mutated putative cation coordinating amino acids in transmembrane hairpin M5-M6 of rat Na,K-ATPase: Asp776 (Gln, Asp, Ala), Glu779 (Asp, Gln, Ala), Asp804 (Glu, Asn, Ala), and Asp808 (Glu, Asn, Ala). Electrogenic cation transport properties of these 12 mutants were analyzed in two-electrode voltage-clamp experiments on Xenopus laevis oocytes by measuring the voltage dependence of K+-stimulated stationary currents and pre-steady-state currents under electrogenic Na+/Na+ exchange conditions. Whereas mutants D804N, D804A, and D808A hardly showed any Na+/K+ pump currents, the other constructs could be classified according to the [K+] and voltage dependence of their stationary currents; mutants N776A and E779Q behaved similarly to the wild-type enzyme. Mutants E779D, E779A, D808E, and D808N had in common a decreased apparent affinity for extracellular K+. Mutants N776Q, N776D, and D804E showed large deviations from the wild-type behavior; the currents generated by mutant N776D showed weaker voltage dependence, and the current-voltage curves of mutants N776Q and D804E exhibited a negative slope. The apparent rate constants determined from transient Na+/Na+ exchange currents are rather voltage-independent and at potentials above -60 mV faster than the wild type. Thus, the characteristic voltage-dependent increase of the rate constants at hyperpolarizing potentials is almost absent in these mutants. Accordingly, dislocating the carboxamide or carboxyl group of Asn776 and Asp804, respectively, decreases the extracellular Na+ affinity.

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Language(s): eng - English
 Dates: 2003-09-292003-06-172021-01-042003-12-19
 Publication Status: Published in print
 Pages: 10
 Publishing info: -
 Table of Contents: -
 Rev. Type: Peer
 Identifiers: DOI: 10.1074/jbc.M306384200
PMID: 14532287
 Degree: -

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Title: The Journal of Biological Chemistry
  Other : JBC
Source Genre: Journal
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Publ. Info: Baltimore, etc. : American Society for Biochemistry and Molecular Biology [etc.]
Pages: - Volume / Issue: 278 (51) Sequence Number: - Start / End Page: 51213 - 51222 Identifier: ISSN: 0021-9258
CoNE: https://pure.mpg.de/cone/journals/resource/954925410826_1