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Packing of the Transmembrane Helices of Na,K-ATPase:  Direct Contact between β-Subunit and H8 Segment of α-Subunit Revealed by Oxidative Cross-Linking

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Department of Pharmacology, Medical College of Ohio, Toledo, Ohio 43614 USA
Cite this: Biochemistry 2000, 39, 32, 9778–9785
Publication Date (Web):July 14, 2000
https://doi.org/10.1021/bi001004j
Copyright © 2000 American Chemical Society

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    Abstract

    Spatial relationships among the transmembrane (TM) segments of α- and β-subunits of the Na,K-ATPase molecule have been investigated using oxidative induction of disulfide bonds. The catalytic α-subunit contains 10 TM α-helices (H1−H10) with 9 Cys residues located within or close to the membrane moiety. There is one Cys residue in the single TM segment of β-subunit (Hβ). Previously, the cross-linking products containing the β-subunit and two fragments of α-subunit (the N-terminal containing H1−H2 helices and the C-terminal containing H7−H10 helices) have been identified in experiments with membrane-bound or detergent-solubilized preparations of the membrane moiety of trypsin-digested Na,K-ATPase [Sarvazyan, N. A., Modyanov, N. N., and Askari, A. (1995) J. Biol. Chem. 270, 26528−26532 and Sarvazyan, N. A., Ivanov, A., Modyanov, N. N., and Askari, A. (1997) J. Biol. Chem. 272, 7855−7858]. Here, we have shown that Cu2+-phenanthroline treatment of digitonin-solubilized preparation provides the most efficient formation of intersubunit cross-linked product that is predominantly a dimer of β-subunit and a 22-kDa C-terminal α-fragment containing H7−H10 helices. This cross-linked product was isolated and subjected to CNBr cleavage. The resulting fragments were electrophoretically separated and sequenced. A 17-kDa peptide composed of Ile853−Met942 α-fragment and Ala5−Met56 β-fragment was identified as a product of intersubunit disulfide cross-link between Cys44 of Hβ and either Cys911 or Cys930, located in H8. This provides the first direct experimental evidence of the juxtaposition of Hβ and H8 within the Na,K-ATPase molecule. The second detected cross-linked product was composed of α-fragments Lys947−Met963 and Tyr974−Tyr1016 linked by induced disulfide bridge between Cys964 (H9) and Cys983 (H10). The spatial proximity of these Cys residues defines the mutual orientation of H9 and H10 helices of α-subunit.

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     This work was supported by National Institutes of Health Grants GM54997 and HL-36573.

    *

     To whom correspondence should be addressed at Department of Pharmacology, Medical College of Ohio, 3035 Arlington Avenue, Toledo, OH 43614-5804. Telephone:  (419) 383-4182. Fax:  (419) 383-2871. E-mail:  [email protected].

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    2. Kai Y. Xu. Allosteric property of the (Na++K+)-ATPase β1 subunit. Biochemical and Biophysical Research Communications 2011, 415 (3) , 479-484. https://doi.org/10.1016/j.bbrc.2011.10.098
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    6. Mariana Oana Popa, Holger Lerche. Cu 2+ (1,10 phenanthroline) 3 is an open‐channel blocker of the human skeletal muscle sodium channel. British Journal of Pharmacology 2006, 147 (7) , 808-814. https://doi.org/10.1038/sj.bjp.0706667
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    8. Melissa D. Laughery, Matthew L. Todd, Jack H. Kaplan. Mutational Analysis of α-β Subunit Interactions in the Delivery of Na,K-ATPase Heterodimers to the Plasma Membrane. Journal of Biological Chemistry 2003, 278 (37) , 34794-34803. https://doi.org/10.1074/jbc.M302899200
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    10. CIMING LI, GILLES CRAMBERT, UDO HASLER, KÄTHI GEERING. Na,K‐ATPase α‐β Subunit Interactions in the Transmembrane Domain. Annals of the New York Academy of Sciences 2003, 986 (1) , 226-228. https://doi.org/10.1111/j.1749-6632.2003.tb07165.x
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    14. Udo Hasler, Gilles Crambert, Jean-Daniel Horisberger, Käthi Geering. Structural and Functional Features of the Transmembrane Domain of the Na,K-ATPase % Subunit Revealed by Tryptophan Scanning. Journal of Biological Chemistry 2001, 276 (19) , 16356-16364. https://doi.org/10.1074/jbc.M008778200
    15. Kathleen J. Sweadner, Marina S. Feschenko. Predicted location and limited accessibility of protein kinase A phosphorylation site on Na-K-ATPase. American Journal of Physiology-Cell Physiology 2001, 280 (4) , C1017-C1026. https://doi.org/10.1152/ajpcell.2001.280.4.C1017
    16. Claudia Donnet, Elena Arystarkhova, Kathleen J. Sweadner. Thermal Denaturation of the Na,K-ATPase Provides Evidence for α-α Oligomeric Interaction and γ Subunit Association with the C-terminal Domain. Journal of Biological Chemistry 2001, 276 (10) , 7357-7365. https://doi.org/10.1074/jbc.M009131200

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