Structural Modeling and Electron Paramagnetic Resonance Spectroscopy of the Human Na+/H+ Exchanger Isoform 1, NHE1

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We previously presented evidence that transmembrane domain (TM) IV and TM X-XI are important for inhibitor binding and ion transport by the human Na+/H+ exchanger, hNHE1 (Pedersen, S. F., King, S. A., Nygaard, E. B., Rigor, R. R., and Cala, P. M. (2007) J. Biol. Chem. 282, 19716-19727). Here, we present a structural model of the transmembrane part of hNHE1 that further supports this conclusion. The hNHE1 model was based on the crystal structure of the Escherichia coli Na+/H+ antiporter, NhaA, and previous cysteine scanning accessibility studies of hNHE1 and was validated by EPR spectroscopy of spin labels in TM IV and TM XI, as well as by functional analysis of hNHE1 mutants. Removal of all endogenous cysteines in hNHE1, introduction of the mutations A173C (TM IV) and/or I461C (TM XI), and expression of the constructs in mammalian cells resulted in functional hNHE1 proteins. The distance between these spin labels was similar to 15 A, confirming that TM IV and TM XI are in close proximity. This distance was decreased both at pH 5.1 and in the presence of the NHE1 inhibitor cariporide. A similar TM IV.TM XI distance and a similar change upon a pH shift were found for the cariporide-insensitive Pleuronectes americanus (pa) NHE1; however, in paNHE1, cariporide had no effect on TM IV.TM XI distance. The central role of the TM IV.TM XI arrangement was confirmed by the partial loss of function upon mutation of Arg(425), which the model predicts stabilizes this arrangement. The data are consistent with a role for TM IV and TM XI rearrangements coincident with ion translocation and inhibitor binding by hNHE1.


  • Eva B. Nygaard
  • Jens Lagerstedt
  • Gabriel Bjerre
  • Biao Shi
  • Madhu Budamagunta
  • Kristian A. Poulsen
  • Stine Meinild
  • Robert R. Rigor
  • John C. Voss
  • Peter M. Cala
  • Stine F. Pedersen
Research areas and keywords

Subject classification (UKÄ) – MANDATORY

  • Cell and Molecular Biology
Original languageEnglish
Pages (from-to)634-648
JournalJournal of Biological Chemistry
Issue number1
Publication statusPublished - 2011
Publication categoryResearch

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