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The liquid-ordered phase in sphingomyelincholesterol membranes as detected by the discrimination by oxygen transport (DOT) method

Cellular & Molecular Biology Letters volume 13, pages 430–451 (2008)Cite this article

Abstract

Membranes made from binary mixtures of egg sphingomyelin (ESM) and cholesterol were investigated using conventional and saturation-recovery EPR observations of the 5-doxylstearic acid spin label (5-SASL). The effects of cholesterol on membrane order and the oxygen transport parameter (bimolecular collision rate of molecular oxygen with the nitroxide spin label) were monitored at the depth of the fifth carbon in fluid- and gel-phase ESM membranes. The saturation-recovery EPR discrimination by oxygen transport (DOT) method allowed the discrimination of the liquid-ordered (l o), liquid-disordered (l d), and solid-ordered (s o) phases because the bimolecular collision rates of the molecular oxygen with the nitroxide spin label differ in these phases. Additionally, oxygen collision rates (the oxygen transport parameter) were obtained in coexisting phases without the need for their separation, which provides information about the internal dynamics of each phase. The addition of cholesterol causes a dramatic decrease in the oxygen transport parameter around the nitroxide moiety of 5-SASL in the l o phase, which at 50 mol% cholesterol becomes ∼5 times smaller than in the pure ESM membrane in the l d phase, and ∼2 times smaller than in the pure ESM membrane in the s o phase. The overall change in the oxygen transport parameter is as large as ∼20-fold. Conventional EPR spectra show that 5-SASL is maximally immobilized at the phase boundary between regions with coexisting l d and l o phases or s o and l o phases and the region with a single l o phase. The obtained results all owed for the construction of a phase diagram for the ESM-cholesterol membrane.

Abbreviations

BSM:

bovine brain sphingomyelin

DMPC:

dimyristoylphosphatidylcholine

DPPC:

dipalmitoylphosphatidylcholine

EPR:

electron paramagnetic resonance

ESM:

egg sphingomyelin

FOT:

fast oxygen transport

l d :

liquid-disordered

l o :

liquid-ordered

PC:

phosphatidylcholine

PSM:

palmitoylsphingomyelin

5-SASL:

5-doxylstearic acid spin label

SLOT:

slow oxygen transport

s o :

solid-ordered

T 1 :

spin-lattice relaxation time

References

  1. Simons, K. and Ikonen, E. Functional rafts in cell membranes. Nature 387 (1997) 569–572.

    Article  PubMed  CAS  Google Scholar 

  2. Shaikh, S.R. and Edidin, M.A. Membranes are not just rafts. Chem. Phys. Lipids 144 (2006) 1–3.

    PubMed  CAS  Google Scholar 

  3. Kusumi, A., Koyama-Honda, I. and Suzuki, K. Molecular dynamics and interactions for creation of stimulation-induced stabilized rafts from small unstable steady-state rafts. Traffic 5 (2004) 213–230.

    Article  PubMed  CAS  Google Scholar 

  4. Mayor, S. and Rao, M. Rafts, scale dependent, active lipid organization at the cell surface. Traffic 5 (2004) 231–240.

    Article  PubMed  CAS  Google Scholar 

  5. Edidin, M. The state of lipid rafts from model membranes to cells. Annu. Rev. Biophys. Biomol. Struct. 32 (2003) 257–283.

    Article  PubMed  CAS  Google Scholar 

  6. Munro, S. Lipid rafts: delusive or illusive. Cell 115 (2003) 377–388.

    Article  PubMed  CAS  Google Scholar 

  7. Brown, D.A. and London, E. Structure and function of sphingolipid-and cholesterol-rich membrane rafts. J. Biol. Chem. 275 (2000) 17221–17224.

    Article  PubMed  CAS  Google Scholar 

  8. Brown, D.A. and Rose, J.K. Sorting of GPI-anchored proteins to glycolipidenriched membrane subdomains during transport to the apical cell surface. Cell 68 (1992) 533–544.

    Article  PubMed  CAS  Google Scholar 

  9. Ridgway, N.D. Interaction between metabolism and intracellular distribution of cholesterol and sphingomyelin. Biochim. Biophys. Acta 1484 (2000) 129–141.

    PubMed  CAS  Google Scholar 

  10. Pralle, A., Keller, P., Florin, E.L., Simons, K. and Horber, J.K.H. Sphingolipid-cholesterol rafts diffuse as small entities in the plasma membrane of mammalian cells. J. Cell Biol. 148 (2000) 997–1008.

    Article  PubMed  CAS  Google Scholar 

  11. Friedrichson, T. and Kurzchalia, T.V. Microdomains of GPI-anchored proteins in living cells revealed by crosslinking. Nature 394 (1998) 802–805.

    Article  PubMed  CAS  Google Scholar 

  12. Heerklotz, H. Triton promotes domain formation in lipid raft mixtures. Biophys. J. 83 (2002) 2693–2701.

    PubMed  CAS  Google Scholar 

  13. Pike, L.J. Rafts defined: a report on the Keystone symposium on lipid rafts and cell function. J. Lipid Res. 47 (2006) 1597–1598.

    Article  PubMed  CAS  Google Scholar 

  14. Kawasaki, K., Yin, J.-J., Subczynski, W.K., Hyde, J.S. and Kusumi. A. Pulse EPR detection of lipid exchange between protein-rich raft and bulk domains in the membrane: Methodology development and its application to studies of influenza viral membrane. Biophys. J. 80 (2001) 738–748.

    PubMed  CAS  Google Scholar 

  15. Suzuki, K.G.N., Fujiwara, T.K., Sanematsu, F., Iino, R., Edidin, M. and Kusumi, A. GPI-anchored receptor clusters transiently recruit Lyn and Ga for temporary cluster immobilization and Lyn activation: single-molecule tracking study 1. J. Cell Biol. 177 (2007) 717–730.

    Article  PubMed  CAS  Google Scholar 

  16. Suzuki, K.G.N., Fujiwara, T.K., Edidin, M. and Kusumi, A. Dynamic recruitment of phospholipase Cγ at transiently immobilized GPI-anchored receptor clusters induces IP3−Ca+2 signaling: single-molecule tracking study 2. J. Cell Biol. 177 (2007) 731–742.

    Article  PubMed  CAS  Google Scholar 

  17. Kusumi, A., Subczynski, W.K., Pasenkiewicz-Gierula, M., Hyde, J.S. and Merkle H. Spin-label studies on phosphatidylcholine-cholesterol membranes: effects of alkyl chain length and unsaturation in the fluid phase. Biochim. Biophys. Acta 854 (1986) 307–317.

    Article  PubMed  CAS  Google Scholar 

  18. Recktenwald, D.J. and McConnell, H.M. Phase equilibria in binary mixture of phosphatidylcholine and cholesterol. Biochemistry 20 (1981) 4505–4510.

    Article  PubMed  CAS  Google Scholar 

  19. Shimshick, E.J. and McConnell, H.M. Lateral phase separation in phospholipid membranes. Biochemistry 12 (1973) 2351–2360.

    Article  PubMed  CAS  Google Scholar 

  20. Subczynski, W.K., Widomska, J., Wisniewska, A. and Kusumi, A. Saturation recovery EPR discrimination by oxygen transport (DOT) method for characterizing membrane domains. in: Methods in Molecular Biology. Vol. 398. Lipid Rafts. (McIntosh, T.J., Ed.), Humana Press Inc., Totowa, New York, 2007, 145–160.

    Google Scholar 

  21. Ashikawa, I., Yin, J.-J., Subczynski, W.K., Kouyama, T., Hyde, J.S. and Kusumi. A. Molecular organization and dynamics in bacteriorhodopsin-rich reconstituted membranes: Discrimination of lipid environments by the oxygen transport parameter using a pulse ESR spin-labeling technique. Biochemistry 33 (1994) 4947–4952.

    Article  PubMed  CAS  Google Scholar 

  22. Subczynski, W.K., Wisniewska, A., Hyde, J.S. and Kusumi, A. Three-dimensional dynamic structure of the liquid-ordered domain as examined by a pulse-EPR oxygen probing. Biophys. J. 92 (2007) 1573–1584.

    Article  PubMed  CAS  Google Scholar 

  23. Subczynski, W.K., Pasenkiewicz-Gierula, M., McElhaney, R.N., Hyde, J.S. and Kusumi, A. Molecular dynamics of 1-palmitoyl-2-oleoylphosphatidylcholine membranes containing transmembrane α-helical peptides with alternating leucine and alanine residues. Biochemistry 42 (2003) 3939–3948.

    Article  PubMed  CAS  Google Scholar 

  24. Subczynski, W.K., Lewis, R.N.A.H., McElhaney, R.N., Hodges, R.S., Hyde, J.S. and Kusumi, A. Molecular organization and dynamics of 1-palmitoyl-2-oleoylphosphatidylcholine bilayers containing a transmembrane α-helical peptide. Biochemistry 37 (1998) 3156–3164.

    Article  PubMed  CAS  Google Scholar 

  25. Subczynski, W.K., Hopwood, L.E. and Hyde, J.S. Is the mammalian cell plasma membrane a barrier to oxygen transport? J. Gen. Physiol. 100 (1992) 69–87.

    Article  PubMed  CAS  Google Scholar 

  26. Subczynski, W.K., Hyde, J.S. and Kusumi, A. Effect of alkyl chain unsaturation and cholesterol intercalation on oxygen transport in membranes: a pulse ESR spin labeling study. Biochemistry 30 (1991) 8578–8590.

    Article  PubMed  CAS  Google Scholar 

  27. Subczynski, W.K., Hyde, J.S. and Kusumi, A. Oxygen permeability of phosphatidylcholine-cholesterol membranes. Proc. Natl. Acad. Sci. USA 86 (1989) 4474–4478.

    Article  PubMed  CAS  Google Scholar 

  28. Kusumi, A., Subczynski, W.K. and Hyde, J.S. Oxygen transport parameter in membranes as deduced by saturation recovery measurements of spinlattice relaxation times of spin labels. Proc. Natl. Acad. Sci. USA 79 (1982) 1854–1858.

    Article  PubMed  CAS  Google Scholar 

  29. Ge, M., Field, K.A., Aneja, R., Holovka, D., Baird, B. and Freed J.H. Electron spin resonance characterization of liquid ordered phase of detergent-resistant membranes from RBL-2H3 cells. Biophys. J. 77 (1999) 925–933.

    Article  PubMed  CAS  Google Scholar 

  30. London, E. Insights into lipid raft structure and formation from experiments in model systems. Curr. Opin. Struct. Biol. 12 (2002) 480–486.

    Article  PubMed  CAS  Google Scholar 

  31. Almeida, P.F.F., Vaz, W.L.C. and Thompson, T.E. Lateral diffusion in the liquid phases of dimyristoylphosphatidylcholine/cholesterol bilayers: a free volume analysis. Biochemistry 31 (1992) 6739–6747.

    Article  PubMed  CAS  Google Scholar 

  32. Costa-Filho, A.J., Shimoyama, Y. and Freed, J.H. A2D-ELDOR study of the liquid ordered phase in multilamellar vesicle membranes. Biophys. J. 84 (2003) 2619–2633.

    Article  PubMed  CAS  Google Scholar 

  33. Ge, M., Gidvani, A., Brown, H.A., Holovka, D., Baird, B. and Freed, J.H. Ordered and disordered phases coexist in plasma membrane vesicles of RBL-2H3 mast cells. Biophys. J. 85 (2003) 1278–1288.

    PubMed  CAS  Google Scholar 

  34. Veiga, M.P., Arrondo, J.L.R., Goni, F.M., Alonso, A. and Marsh, D. Interaction of cholesterol with sphingomyelin in mixed membranes containing phosphatidylcholine, studied by spin-label ESR and IR spectroscopies. A possible stabilization of gel-phase sphingolipid domains by cholesterol. Biochemistry 40 (2001) 2614–1622.

    Article  PubMed  CAS  Google Scholar 

  35. Wolf, C. and Chachaty, C. Compared effects of cholesterol and 7-dehydrocholesterol on sphingomyelin-glycerophospholipid bilayers studied by ESR. Biophys. Chem. 84 (2000) 269–279.

    Article  PubMed  CAS  Google Scholar 

  36. Gaffney, B.J. and Marsh, D. High-frequency, spin-label EPR of nonaxial lipid ordering and motion in cholesterol-containing membranes. Proc. Natl. Acad. Sci. USA 95 (1998) 12490–12493.

    Google Scholar 

  37. Almeida, P.F.F., Pokorny, A. and Hinderliter, A. Thermodynamics of membrane domains. Biochim. Biophys. Acta 1720 (2005) 1–13.

    Article  PubMed  CAS  Google Scholar 

  38. Sankaram, M.B. and Thompson, T.E. Interaction of cholesterol with various glycerophospholipids and sphingomyelin. Biochemistry 29 (1990) 10670–10675.

    Article  PubMed  CAS  Google Scholar 

  39. de Almeida, R.F.M., Fedorov, A. and Prieto, M. Sphingomyelin/phosphatidylcholine/cholesterol phase diagram: boundaries and composition of lipid rafts. Biophys. J. 85 (2003) 2406–2416.

    PubMed  Google Scholar 

  40. Kusumi, A., Subczynski, W.K. and Hyde, J.S. Effects of pH on ESR spectra of stearic acid spin labels in membranes: probing the membrane surface. Fed. Proc. 41 (1982) 1394.

    Google Scholar 

  41. Papahadjopoulos, D. Surface properties of acidic phospholipids: Interaction of monolayers and hydrated liquid crystals with uni-and bi-valent metal ions. Biochim. Biophys. Acta 163 (1968) 240–254.

    Article  PubMed  CAS  Google Scholar 

  42. Hyde, J.S. and Subczynski, W.K. Spin-label oximetry. in: Biological Magnetic Resonance. Vol. 8. Spin labeling: theory and applications. (Berliner, L.J. and Reuben, J. Eds.), Plenum, New York, 1989, 399–425.

    Google Scholar 

  43. Subczynski, W.K., Felix, C.C., Klug, C.S. and Hyde, J.S. Concentration by centrifugation for gas exchange EPR oximetry measurements with loop-gap resonators. J. Magn. Reson. 176 (2005) 244–248.

    Article  PubMed  CAS  Google Scholar 

  44. Yin, J.J. and Subczynski, W.K. Effect of lutein and cholesterol on alkyl chain bending in lipid bilayers: a pulse electron paramagnetic resonance spin labeling study. Biophys. J. 71 (1996) 832–839.

    PubMed  CAS  Google Scholar 

  45. Yin, J.J., Pasenkiewicz-Gierula, M. and Hyde, J.S. Lateral diffusion of lipids in membranes by pulse saturation recovery electron spin resonance. Proc. Natl. Acad. Sci. USA 84 (1987) 964–968.

    Article  PubMed  CAS  Google Scholar 

  46. Windrem, D.A. and Plachy, W.Z. The diffusion-solubility of oxygen in lipid bilayers. Biochim. Biophys. Acta 600 (1980) 655–665.

    Article  PubMed  CAS  Google Scholar 

  47. Robinson, B.H., Hass, D.A. and Mailer, C. Molecular dynamics in liquid: spin lattice relaxation of nitroxide spin labels. Science 263 (1994) 490–493.

    Article  PubMed  CAS  Google Scholar 

  48. Loura, L.M.S., Fedorov, A. and Prieto, M. Fluid-fluid membrane microheterogenity: a fluorescence resonance energy transfer study. Biophys. J. 80 (2001) 778–788.

    Article  Google Scholar 

  49. Subczynski, W.K. and Kusumi, A. Effects of very small amounts of cholesterol on gel-phase phosphatidylcholine membranes. Biochim. Biophys. Acta 854 (1986) 318–320.

    Article  PubMed  CAS  Google Scholar 

  50. Widomska, J., Raguz, M., Dillon, J., Gaillard, E.R. and Subczynski, W.K. Physical properties of the lipid bilayer membrane made of calf lens lipids: EPR spin labeling studies. Biochim. Biophys. Acta 1768 (2007) 1454–1465.

    Article  PubMed  CAS  Google Scholar 

  51. Widomska, J., Raguz, M. and Subczynski, W.K. Oxygen permeability of the lipid bilayer membrane made of calf lens lipids. Biochim. Biophys. Acta 1768 (2007) 2635–2645.

    Article  PubMed  CAS  Google Scholar 

  52. Collado, M.I., Goni, F.M., Alonso, A. and Marsh, D. Domain formation in sphingomyelin/cholesterol mixed membranes studied by spin-label electron spin resonance spectroscopy. Biochemistry 44 (2005) 4911–4918.

    Article  PubMed  CAS  Google Scholar 

  53. Hubbell, W.L. and McConnell, H.M. Molecular motion in spin-labeled phospholipids and membranes. J. Am. Chem. Soc. 93 (1971) 314–326.

    Article  PubMed  CAS  Google Scholar 

  54. Gaffney, B.J. Principal considerations for the calculation of order parameters for fatty acid or phospholipid spin labels in membranes. in: Spin labeling: theory and applications. (Berliner, L.J. Ed.), Academic Press, New York, 1976, 567–571.

    Google Scholar 

  55. Koynova, R. and Caffrey, M. Phases and phase transitions of the sphingolipids. Biochim. Biophys. Acta 1255 (1995) 213–236.

    PubMed  Google Scholar 

  56. Ramstedt, B. and Slotte, J.P. Sphingolipids and the formation of sterolenriched ordered membrane domains. Biochim. Biophys. Acta 1758 (2006) 1945–1956.

    Article  PubMed  CAS  Google Scholar 

  57. Guo, W., Kurze, V., Huber, T., Afdhal, N.H., Beyer, K. and Hamilton, J.A. A solid-state NMR study of phospholipid-cholesterol interactions: sphingomyelin-cholesterol binary systems. Biophys. J. 83 (2002) 1465–1478.

    PubMed  CAS  Google Scholar 

  58. Ohvo, H. and Slotte, J.P. Cyclodextrin-mediated removal of sterols from monolayers: effects of sterol structure and phospholipids on desorption rate. Biochemistry 35 (1996) 8018–8024.

    Article  PubMed  CAS  Google Scholar 

  59. Slotte, J.P. Enzyme-catalyzed oxidation of cholesterol in mixed phospholipid monolayers reveals stoichiometry at which free cholesterol clusters disappear. Biochemistry 31 (1992) 5472–5477.

    Article  PubMed  CAS  Google Scholar 

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Authors and Affiliations

  1. Department of Biophysics, Medical College of Wisconsin, Milwaukee, WI, 53226, USA

    Anna Wisniewska & Witold K. Subczynski

  2. Department of Biophysics, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Krakow, Poland

    Anna Wisniewska

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  1. Anna Wisniewska
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Correspondence to Anna Wisniewska.

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Wisniewska, A., Subczynski, W.K. The liquid-ordered phase in sphingomyelincholesterol membranes as detected by the discrimination by oxygen transport (DOT) method. Cell Mol Biol Lett 13, 430–451 (2008). https://doi.org/10.2478/s11658-008-0012-y

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Cellular & Molecular Biology Letters

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