To Image the Orientation and Spatial Distribution of Reconstituted Na+,K+-ATPase in Model Lipid Membranes
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.
KeywordsNa+,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.
- 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
- 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.J.C. Skou, M. Esmann, The Na,K-ATPase. J. Bioenerg. Biomembr. 24(3), 249–261 (1992)Google Scholar
- 15.H.O. Schatzman, Herzglycoside und Kationentransport. Helv. Physiol. Pharmacol. Acta 11, 346 (1953)Google Scholar
- 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
- 23.O.H. Lowry, Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265–275 (1951)Google Scholar
- 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
- 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). https://doi.org/10.1038/protex.2016.010
- 36.S. Safran, Micelles Membranes Microemulsions and Monolayers (Springer, New York, 1994)Google Scholar