To Image the Orientation and Spatial Distribution of Reconstituted Na+,K+-ATPase in Model Lipid Membranes

  • Tripta BhatiaEmail author
  • Flemming Cornelius


Imaging of sub-optical dynamic features, such as functional membrane nanodomains that are short-lived and proteins that are embedded in such nanodomains, is a challenge with the currently available imaging techniques because such features of interests are very dynamic. Our approach to image dynamic suboptical features is based on a GUV-collapse method followed by high-resolution imaging. We have functionally reconstituted Na+,K+-ATPase, a P-type transmembrane ATPase protein, into free-standing giant unilamellar vesicles (GUVs) which are collapsed to form planar lipid bilayer (PLB) patches within the ∼10 ms time scale. Using our method, we have successfully imaged the PLB patches using atomic force microscopy under physiological conditions to quantify orientation and density of Na+,K+-ATPase in membrane with nanoscopic domains.


Na+,K+-ATPase GUVs Transmembrane protein reconstitution Membrane rafts Immunolabelling AFM 



TB acknowledges Prof. John H. Ipsen, Prof. O.G. Mouritsen, A.C. Simonsen (SDU), P.L. Hansen, L.A. Bagatolli, J. Brewer (SDU), L. Duelund, B. Franchi and H. Kidmose for useful discussions on the work presented in this chapter.


  1. 1.
    R.W. Albers, Biochemical aspects of active transport. Annu. Rev. Biochem. 36, 727–756 (1967)CrossRefGoogle Scholar
  2. 2.
    R.L. Post, C. Hegyvary, S. Kume, Activation by adenosine triphosphate in the phosphorylation kinetics of sodium and potassium ion transport adenosine triphosphatase. J. Biol. Chem. 247, 6530–6540 (1972)Google Scholar
  3. 3.
    R. Kanai, H. Ogawa, B. Vilsen, F. Cornelius, C. Toyoshima, Crystal structure of a Na+-bound Na+,K+-ATPase preceding the E1P state. Nature 502, 201–206 (2013)CrossRefGoogle Scholar
  4. 4.
    T. Shinoda, H. Ogawa, F. Cornelius, C. Toyoshima, Crystal structure of the sodium-potassium pump at 2.4 Å resolution. Nature 459, 446–450 (2009)CrossRefGoogle Scholar
  5. 5.
    H. Ogawa, T. Shinoda, F. Cornelius, C. Toyoshima, Crystal structure of the sodium-potassium pump (Na+,K+-ATPase) with bound potassium and ouabain. Proc. Natl. Acad. Sci. U. S. A. 106(33), 13742–13747 (2009)CrossRefGoogle Scholar
  6. 6.
    H. Ogawa, F. Cornelius, A. Hirata, C. Toyoshima, Sequential substitution of K(+) bound to Na+,K+-ATPase visualized by X-ray crystallography. Nat. Commun. 6, 8004 (2015)CrossRefGoogle Scholar
  7. 7.
    J. Skou, The influence of some cations on an adenosine triphosphatase from peripheral nerves. Biochim. Biophys. Acta 23, 394–401 (1957)CrossRefGoogle Scholar
  8. 8.
    F. Cornelius, In Biomimetic Membranes for Sensor and Separation Applications, Biological and Medical Physics, Biomedical Engineering, ed. by C. Hèlix-Nielsen, vol. 6, (Springer, Springer Netherlands, 2012), p. 113–135. DOI: 10.1007/978-94-007-2184-5Google Scholar
  9. 9.
    J.C. Skou, M. Esmann, The Na,K-ATPase. J. Bioenerg. Biomembr. 24(3), 249–261 (1992)Google Scholar
  10. 10.
    J.D. Robinson, Steps to the Na+,K+ pump and Na+,K+-ATPase (1939–1962). Physiology 10(4), 184 (1995)CrossRefGoogle Scholar
  11. 11.
    F. Cornelius, M. Habeck, R. Kanai, C. Toyoshima, S.J.D. Karlish, General and specific lipid-protein interactions in NaK-ATPase. Biochim. Biophys. Acta 1848, 1729–1743 (2015)CrossRefGoogle Scholar
  12. 12.
    P.L. Jorgensen, K.O. Håkansson, S.J.D. Karlish, Structure and mechanism of Na,K-ATPase: functional sites and their interactions. Annu. Rev. Physiol. 65(1), 817–849 (2003)CrossRefGoogle Scholar
  13. 13.
    J.H. Kaplan, Biochemistry of Na+,K+-ATPase. Annu. Rev. Biochem. 71(1), 511–535 (2002)CrossRefGoogle Scholar
  14. 14.
    W.J. Rice, H.S. Young, D.W. Martin, J.R. Sachs, D.L. Stokes, Structure of Na+,K+-ATPase at 11-Å resolution: comparison with Ca2+-ATPase in E1 and E2 states. Biophys. J. 80(5), 2187–2197 (2001)CrossRefGoogle Scholar
  15. 15.
    H.O. Schatzman, Herzglycoside und Kationentransport. Helv. Physiol. Pharmacol. Acta 11, 346 (1953)Google Scholar
  16. 16.
    D.W. Martin, Structure-function relationships in the Na+,K+-pump, in Seminars in Nephrology, vol. 25, (Elsevier BV Netherlands, 2005), pp. 282–291. DOI: 10.1016/j.semnephrol.2005.03.003Google Scholar
  17. 17.
    Z. Xie, Molecular mechanisms of Na+,K+-ATPase mediated signal transduction. Ann. N. Y. Acad. Sci. 986, 497–503 (2003)CrossRefGoogle Scholar
  18. 18.
    D.W. Martin, J.R. Sachs, Preparation of Na+,K+-ATPase with near maximal specific activity and phosphorylation capacity: evidence that the reaction mechanism involves all of the sites. Biochemistry 38(23), 7485–7497 (1999)CrossRefGoogle Scholar
  19. 19.
    H. Poulsen, H. Khandelia, J.P. Morth, M. Bublitz, O.G. Mouritsen, J. Egebjerg, P. Nissen, Neurological disease mutations compromise a C-terminal ion pathway in the Na+,K+-ATPase. Nature 467(7311), 99–102 (2010)CrossRefGoogle Scholar
  20. 20.
    J.C. Skou, M. Esmann, Preparation of membrane Na+,K+-ATPase from rectal glands of Squalus acanthias. Methods Enzymol. 156, 43–46 (1988)CrossRefGoogle Scholar
  21. 21.
    F. Cornelius, Functional reconstitution of the sodium pump kinetics of exchange reactions performed by reconstituted NaK-ATPase. Biochim. Biophys. Acta 1071, 19–66 (1991)CrossRefGoogle Scholar
  22. 22.
    P. Ottolenghi, The reversible delipidation of a solubilized sodium-plus-potassium ion-dependent adenosine triphosphatase from the salt gland of the spiny dogfish. Biochem. J. 151(1), 61 (1975)CrossRefGoogle Scholar
  23. 23.
    O.H. Lowry, Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265–275 (1951)Google Scholar
  24. 24.
    F. Cornelius, J.V. Moller, In Handbook of Non-medical Applications of Liposomes, ed. by D. D. Lasic, Y. Barenholz, vol. 2, (CRC Press, Boca Raton, 1995), pp. 219–243Google Scholar
  25. 25.
    F. Cornelius, Incorporation of C12E8-solubilized Na+,K+-ATPase into liposomes, determination of sidedness and orientation. Methods Enzymol. 156, 156–167 (1988)CrossRefGoogle Scholar
  26. 26.
    G.L. Peterson, A simplification of the protein assay method of Lowry et al. which is more generally applicable. Anal. Biochem. 83, 346–356 (1977)CrossRefGoogle Scholar
  27. 27.
    T. Bhatia, F. Cornelius, J.H. Ipsen, Capturing sub-optical dynamic structures in the lipid bilayer patches formed from free-standing giant unilamellar vesicles. Nat. Protoc. 12, 1563–1575 (2017)CrossRefGoogle Scholar
  28. 28.
    T. Bhatia, F. Cornelius, O.G. Mouritsen, J.H. Ipsen, Reconstitution of transmembrane protein Na+,K+-ATPase in giant unilamellar vesicles of lipid mixtures involving PSM, DOPC, DPPC and cholesterol at physiological buffer and temperature conditions. Protoc. Exchange. (2016).
  29. 29.
    T. Bhatia et al., Spatial distribution and activity of Na+,K+-ATPase in lipid bilayer membranes with phase boundaries. Biochim. Biophys. Acta 1858, 1390–1399 (2016)CrossRefGoogle Scholar
  30. 30.
    T. Bhatia et al., Preparing giant unilamellar vesicles of complex lipid mixtures on demand: mixing small unilamellar vesicles of compositionally heterogeneous mixtures. Biochim. Biophys. Acta 1848, 3175–3180 (2015)CrossRefGoogle Scholar
  31. 31.
    T. Bhatia, P. Husen, J.H. Ipsen, L.A. Bagatolli, A.C. Simonsen, Fluid domain patterns in free-standing membranes captured on a solid support. Biochim. Biophys. Acta 1838, 2503–2510 (2014)CrossRefGoogle Scholar
  32. 32.
    C. Hamai, P.S. Cremer, S.M. Musser, Single giant vesicle rupture events reveal multiple mechanisms of glass-supported bilayer formation. Biophys. J. 92, 1988–1999 (2007)CrossRefGoogle Scholar
  33. 33.
    D.J. Muller, A. Engel, Atomic force microscopy and spectroscopy of native membrane proteins. Nat. Protoc. 2, 2191–2197 (2007)CrossRefGoogle Scholar
  34. 34.
    F. Jarai-Szabo, Z. Neda, On the size distribution of Poisson Voronoi cells. Physica A 385, 518–526 (2007)CrossRefGoogle Scholar
  35. 35.
    F. Cornelius, Cholesterol dependent interaction of polyunsaturated phospholipids with NaK-ATPase. Biochemistry 47, 1652–1658 (2008)CrossRefGoogle Scholar
  36. 36.
    S. Safran, Micelles Membranes Microemulsions and Monolayers (Springer, New York, 1994)Google Scholar
  37. 37.
    J.V. Møller et al., Probing of the membrane topology of sarcoplasmic reticulum Ca2+-ATPase with sequence-specific antibodies. J. Biol. Chem. 272, 29015–29032 (1997)CrossRefGoogle Scholar
  38. 38.
    C. Toyoshima et al., Crystal structures of the calcium pump and sarcolipin in the Mg+2 -bound E1 state. Nature 495, 260–264 (2013)CrossRefGoogle Scholar
  39. 39.
    J.P. Morth et al., A structural overview of the plasma membrane Na+,K+-ATPase and H+ ATPase ion pumps. Nat. Rev. Mol. Cell Biol. 12, 60–70 (2011)CrossRefGoogle Scholar

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© Springer Nature Switzerland AG 2019

Authors and Affiliations

  1. 1.Max Planck Institute of Colloids and InterfacesGolmGermany
  2. 2.Department of BiomedicineAarhus UniversityAarhusDenmark

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