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

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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.

  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.

  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.

  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.

  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.

  6. 6.

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

  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.

  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.

  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.

  10. 10.

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

  11. 11.

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

  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.

  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.

  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.

  15. 15.

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

  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.

  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.

  18. 18.

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

  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.

  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.

  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.

  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

  23. 23.

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

  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.

  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.

  26. 26.

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

  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.

  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

  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.

  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.

  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.

  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.

  33. 33.

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

  34. 34.

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

  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.

  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.

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Correspondence to H. Duclohier or S. Ladha.

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Keywords

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