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Coupling optical and electrical measurements in artificial membranes: Lateral diffusion of lipids and channel forming peptides in planar bilayers

Abstract

Planar lipid bilayers (PLB) were prepared by the Montal-Mueller technique in a FRAP system designed to simultaneously measure conductivity across, and lateral diffusion of, the bilayer. In the first stage of the project the FRAP system was used to characterise the lateral dynamics of bilayer lipids with regards to phospholipid composition (headgroup, chain unsaturation etc.), presence of cholesterol and the effect of divalent cations on negatively-charged bilayers. In the second stage of the project, lateral diffusion of two fluorescently-labelled voltage-dependent pore-forming peptides (alamethicin and S4s from Shaker K+ channel) was determined at rest and in the conducting state. This study demonstrates the feasibility of such experiments with PLBs, amenable to physical constraints, and thus offers new opportunities for systematic studies of structure-function relationships in membrane-associating molecules.

References

  1. 1.

    Macdonald A.G. and P.C. Wraight 1995. Combined spectroscopic and electrical recording techniques in membrane research: prospects for single channel studies. Prog. Biophys. Molec. Biol. 63, 1–29.

    Article  CAS  Google Scholar 

  2. 2.

    Cohen, L.B., Landowne, D. and B.M. Salzberg. 1990. Optical measurements on squid axons. In “Squid as experimental animals” (edited by D.L. Gilbert, W.J. Adelman, Jr. and J.M. Arnold). Plenum Press, New York. pp 161–170.

    Google Scholar 

  3. 3.

    Shinitzky, M. 1984a. Membrane fluidity and cellular functions. In Physiology of Membrane Fluidity, Vol. I. M. Shinitzky, editor. CRC Press, Inc., Boca Raton, Florida.

    Google Scholar 

  4. 4.

    Cossins, A. R. 1983. The adaptation of membrane structure and function to changes in temperature. In Cellular Adaptation to Environmental Changes. A.R. Cossins and P. Sheterline, editors. Cambridge University Press, Cambridge, London, New York. 3–32.

    Google Scholar 

  5. 5.

    Cuculescu, M., D. Hyde and K. Bowler 1995. Temperature acclimation of marine crabs: changes in plasma membrane fluidity and lipid composition. J. Therm. Biol. 20, 207–222.

    Article  Google Scholar 

  6. 6.

    Shinitzky, M. 1984b. Membrane fluidity in malignancy: adversative and recuperative. Biochim. Biophys. Acta 738, 251–261.

    PubMed  CAS  Google Scholar 

  7. 7.

    Georgescauld, D. and H. Duclohier. 1978. Transient fluorescence signals from pyrene labeled pike nerves during action potentials. Possible implications for membrane fluidity changes. Biochem. Biophys. Res. Commun. 83, 1186–1191.

    Article  Google Scholar 

  8. 8.

    Tocanne, J. F., L. Dupou-Cézanne and A. Lopez. 1994. Lateral diffusion of lipids in model and natural membranes. Prog. Lipid Res. 33, 203–237.

    PubMed  Article  CAS  Google Scholar 

  9. 9.

    Blackwell, M., C. Gubas, S. Gygax, D. Roman and B. Wagner. 1994. The planoquinone diffusion coefficient in chloroplasts and its mechanistic implications. Biochim. Biophys. Acta 1183, 553–543.

    Article  Google Scholar 

  10. 10.

    Lamb, T. D. 1994. Stochastic simulation of activation in the G-protein cascade of phototransduction. Biophys. J. 67, 1439–1454.

    PubMed  Article  CAS  Google Scholar 

  11. 11.

    Schlessinger, J. 1993. Lateral and rotational diffusion of EGF-receptor complex relationship to receptor mediated endocytosis. Biopolymers 22, 47–353

    Google Scholar 

  12. 12.

    Leckband, D. E., J. N. Israelachvili, F. J. Schmitt and W. Knoll. 1992. Long-range attraction and molecular-rearrangements in receptor-ligand interactions. Science 255, 1419–1421.

    PubMed  Article  CAS  Google Scholar 

  13. 13.

    Anderson, C. M., G. N. Georgiou, I. E. G. Morrison, G. V. W. Stevenson and R. J. Cherry. 1992. Tracking of cell surface receptors by fluorescence digital imaging microscopy using a charge coupled device camera — low density lipoprotein and influenza virus receptor mobility at 4°C. J.Cell Sci. 101, 415–425.

    PubMed  Google Scholar 

  14. 14.

    Cherry, R. J., G. N. Georgiou and I. E. G. Morrison. 1994. New insights into the structure of cell membranes from single particle tracking experiments. Biochem. Soc. Trans. 22, 781–784.

    PubMed  CAS  Google Scholar 

  15. 15.

    Jacobson, K., Sheets, E.R. and Simpson, R. 1995. Revisiting the fluid mosaic model. Science 268, 1441–1442.

    PubMed  Article  CAS  Google Scholar 

  16. 16.

    Tournier, J. F., A. Lopez, N. Gas and J. F. Tocanne. 1989. The lateral motion in the apical plasma membrane of endothelial cells is reversibly affected by the presence of cell junctions. Exp. Cell Res. 181, 375–384.

    PubMed  Article  CAS  Google Scholar 

  17. 17.

    Joe, E.H. and Angelides, K.J. 1993. Clustering and mobility of voltage-dependent sodium channels during myelination. J. Neurosci. 13, 2993–3005.

    PubMed  CAS  Google Scholar 

  18. 18.

    Bloom, J. A. and W. W. Webb. 1983. Lipid diffusibility in the intact erythrocyte membrane. Biophys. J. 42, 295–305.

    PubMed  Article  CAS  Google Scholar 

  19. 19.

    Smith, B. A. and H. M. McConnell. 1978. Determination of molecular motion in membranes using periodic pattern photobleaching. Proc. Natl. Acad. Sci. 75, 2759–63.

    PubMed  Article  CAS  Google Scholar 

  20. 20.

    Davoust, J., P. F. Devaux and L. Leger. 1982. Fringe pattern photobleaching, a new method for the measurement of transport coefficients of biological macromolecules. EMBO J. 1, 1233–1238.

    PubMed  CAS  Google Scholar 

  21. 21.

    Wedekind, P., U. Kubitschek, and R. Peters. 1994. Scanning microphotolysis: a new photobleaching technique based on fast intensity modulation of a scanned laser beam and confocal imaging. J. Microsc. 176, 23–33.

    PubMed  CAS  Google Scholar 

  22. 22.

    Krueger, B. K., J. F. Worley and R. J. French. 1983. Single sodium channel from rat brain incorporated into planar lipid bilayers. Nature 303, 172–175

    PubMed  Article  CAS  Google Scholar 

  23. 23.

    Hanke, W. and W. R. Schlue. 1993. Planar lipid bilayers. Methods and applications. Academic Press. London, San Diego.

    Google Scholar 

  24. 24.

    Chang, H. M., R. Reitsjetter, R. P. Mason and R. Gruener. 1995. Attenuation of channel kinetics and conductance by cholesterol: an interpretation using structural stress as a unifying concept. J. Membrane Biol. 143, 51–63.

    Article  CAS  Google Scholar 

  25. 25.

    Koppel, D.E., Axelrod, D., Schlessinger, J., Elson, E.L. and W.W. Webb. 1976. Dynamics of fluorescence marker concentration as a probe of mobility. Biophys. J. 16, 1315–1329.

    PubMed  Article  CAS  Google Scholar 

  26. 26.

    Fahey, P. F. and W. W. Webb. 1978. Lateral diffusion in phospholipid bilayer membranes and multilamellar liquid crystals. Biochemistry. 17, 3046–3053.

    PubMed  Article  CAS  Google Scholar 

  27. 27.

    Montal, M. and P. Mueller. 1972. Formation of bimolecular membranes from monolayers and study of their properties. Proc. Natl. Acad. Sci. USA 69, 3561–3566.

    PubMed  Article  CAS  Google Scholar 

  28. 28.

    Helluin, O., Dugast, J-Y, Molle, G., Mackie, A. R., Ladha, S. and Duclohier, H. (1997) Lateral diffusion and conductance properties of a fluorescein-labelled alamethicin in planar lipid bilayers. Biochimica et Biophysica Acta, 1330, 284–292

    PubMed  Article  CAS  Google Scholar 

  29. 29.

    Peled, Z.H., Arkin, I.T., Engelman, D.M. and Y. Shai. 1996. Coassembly of synthetic segments of Shaker K+ channel within phospholipid membranes. Biochemistry 35, 6828–6838.

    Article  Google Scholar 

  30. 30.

    Ladha, S., A. R. Mackie and D. C. Clark. 1994. Cheek cell membrane fluidity measured by fluorescence recovery after photobleaching and steady state anisotropy. J. Membrane Biol., 142, 223–229.

    Article  CAS  Google Scholar 

  31. 31.

    Clark, D. C., R. Dann, A. R. Mackie, J. Mingins, A. C. Pinder, P. W. Purdy, E. J. Russell, L. J. Smith and D. R. Wilson. 1990. Surface diffusion in SDS stabilized thin liquid films. J. Colloid Interf. Sci., 138, 195–206.

    Article  CAS  Google Scholar 

  32. 32.

    Yguerabide, J., J. A. Schmidt and E. E. Yguerabide. 1982. Lateral mobility in membranes as detected by fluorescence recovery after photobleaching. Biophys. J. 39, 69–75.

    Article  Google Scholar 

  33. 33.

    Wolf, D. E. 1989. Designing, building and using a fluorescence recovery after photobleaching instrument. Methods Cell Biol. 30, 271–306.

    PubMed  Article  CAS  Google Scholar 

  34. 34.

    Schindler, H. 1980. Formation of planar bilayers from artificial or native membrane vesicles. FEBS letters 122, 77–79.

    PubMed  Article  CAS  Google Scholar 

  35. 35.

    Mueller, P., Rudin, D. O., Tien, H. T. and Westcott, W. C. 1962. Reconstitution of cell membrane structure in vitro and its transformation into an excitable system. Nature 194, 979–980.

    PubMed  Article  CAS  Google Scholar 

  36. 36.

    Ladha, S., Mackie, A., Harvey, L., Clark, D., Lea, E., Brullemans, M. and Duclohier, H. (1996) Lateral diffusion in planar lipid bilayers. A FRAP investigation of its modulation by lipid composition, cholesterol or alamethicin content and divalent cations. Biohys. J. 71, 1364–1373.

    Article  CAS  Google Scholar 

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Correspondence to H. Duclohier or S. Ladha.

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Duclohier, H., Helluin, O., Lea, E. et al. Coupling optical and electrical measurements in artificial membranes: Lateral diffusion of lipids and channel forming peptides in planar bilayers. Biol Proced Online 1, 81–91 (1998). https://doi.org/10.1251/bpo10

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Keywords

  • Lateral Diffusion
  • Fluorescence Recovery After Photobleaching
  • Fluorescence Recovery
  • Biological Procedure
  • Planar Lipid Bilayer