Characterizing Molecular Mobility and Membrane Interactions of G Protein-Coupled Receptors

  • Vladana VukojevićEmail author
  • Yu Ming
  • Lars Terenius
Part of the Neuromethods book series (NM, volume 60)


Drugs targeting the opioid neurotransmission system have been used for centuries recreationally and for medical purposes. In spite of this vast experience and competence in opioid pharmacotherapy, fine details about the cellular and molecular mechanisms underlying opioid receptor physiology remain unknown. We present here two methods with single-molecule sensitivity, confocal laser scanning microscopy with avalanche photodiode (APD) detectors (APD imaging) and fluorescence correlation spectroscopy (FCS) suitable for nondestructive study of molecular interactions and intracellular transporting processes in living cells. These high-resolution methods provide functional readouts, giving measures of concentration, mobility and affinity, for the investigated molecules and enable us to monitor changes in these properties in living cells in real time. We have used these methods to study early events in opioid receptor activation with specific and nonspecific ligands, and discuss the new insights obtained by these approaches.

Key words

Opioid receptors Opiates Alcohol Protein–lipid interactions APD imaging Fluorescence correlation spectroscopy (FCS) 



The authors acknowledge support from The Knut and Alice Wallenberg Foundation, The Swedish Research Council and The Swedish Brain Foundation.


  1. 1.
    Singer SJ, Nicolson GL (1972) The fluid mosaic model of the structure of cell membranes. Science 175:720–731.PubMedCrossRefGoogle Scholar
  2. 2.
    Allen JA, Halverson-Tamboli RA et al (2007) Lipid raft microdomains and neurotransmitter signalling. Nat Rev Neurosci 8:128–140.PubMedCrossRefGoogle Scholar
  3. 3.
    Pike LJ (2003) Lipid rafts: bringing order to chaos. J Lipid Res 44:655–667.PubMedCrossRefGoogle Scholar
  4. 4.
    Simons K, Toomre D (2000) Lipid rafts and signal transduction. Nat Rev Mol Cell Biol 1:31–39.PubMedCrossRefGoogle Scholar
  5. 5.
    Hanyaloglu AC, von Zastrow M (2008) Regulation of GPCRs by endocytic membrane trafficking and its potential implications. Ann Rev Pharmacol Toxicol 48:537–568.CrossRefGoogle Scholar
  6. 6.
    Hanson MA, Cherezov V, Griffith MT et al (2008) A specific cholesterol binding site is established by the 2.8 A structure of the human beta2-adrenergic receptor. Structure 16:897–905.PubMedCrossRefGoogle Scholar
  7. 7.
    Vukojević V, Heidkamp M, Ming Y et al (2008) Quantitative single-molecule imaging by confocal laser scanning microscopy. Proc Natl Acad Sci USA 105:18176–18181.PubMedCrossRefGoogle Scholar
  8. 8.
    Elson EL (2004) Quick tour of fluorescence correlation spectroscopy from its inception. J Biomed Optics 9:857–864.CrossRefGoogle Scholar
  9. 9.
    Vukojević V, Pramanik A, Yakovleva T et al (2005) Study of molecular events in cells by fluorescence correlation spectroscopy. Cell Mol Life Sci 62:535–550.PubMedCrossRefGoogle Scholar
  10. 10.
    García-Sáez AJ, Schwille P (2007) Single molecule techniques for the study of membrane proteins. Appl Microbiol Biotechnol 76:257–266.PubMedCrossRefGoogle Scholar
  11. 11.
    Vukojević V, Papadopoulos DK, Terenius L et al (2010) Quantitative study of synthetic Hox transcription factor–DNA interactions in live cells. Proc Natl Acad Sci USA 107:4087–4092.PubMedCrossRefGoogle Scholar
  12. 12.
    Terenius L (1973) Stereospecific interaction between narcotic analgesics and a synaptic plasma-membrane fraction of rat cerebral-­cortex. Acta Pharm Toxicol 32:317–320.CrossRefGoogle Scholar
  13. 13.
    Terenius L (1973) Characteristics of the “receptor” for narcotic analgesics in synaptic plasma membrane fraction from rat brain. Acta Pharmacol Toxicol (Copenh) 33:377–384.CrossRefGoogle Scholar
  14. 14.
    Terenius L. (1977) Opioid peptides and opiates differ in receptor selectivity. Psychoneuroendocrinology 2:53–58.PubMedCrossRefGoogle Scholar
  15. 15.
    Gunne LM, Lindström L, Terenius L (1977) Naloxone-induced reversal of schizophrenic hallucinations. J Neural Transm 40:13–19.PubMedCrossRefGoogle Scholar
  16. 16.
    Terenius L. (1978) Endogenous peptides and analgesia. Ann Rev Pharmacol Toxicol 8:189–204.CrossRefGoogle Scholar
  17. 17.
    Vincent SR, Dalsgaard CJ, Schultzberg M et al (1984) Dynorphin-immunoreactive neurons in the autonomic nervous-system. Neurosci 11:973–987.CrossRefGoogle Scholar
  18. 18.
    Nylander I, Hyytia P, Forsander O et al (1994) Differences between alcohol-preferring (AA) and alcohol-avoiding (ANA) rats in the prodynorphin and proenkephalin systems. Alcohol Clin Exp Res 18:1272–1279.PubMedCrossRefGoogle Scholar
  19. 19.
    Kuzmin A, Sandin J, Terenius L et al (2003) Acquisition, expression, and reinstatement of ethanol-induced conditioned place preference in mice: Effects of opioid receptor-like 1 receptor agonists and naloxone. J Pharmacol Exp Ther 304:310–318.PubMedCrossRefGoogle Scholar
  20. 20.
    Vukojevic´ V, Ming Y, D’Addario C et al (2008) Mu-opioid receptor activation in live cells. FASEB J 22:3537–3548.PubMedCrossRefGoogle Scholar
  21. 21.
    Vukojevic´ V, Ming Y, D’Addario C et al (2008) Ethanol/naltrexone interactions at the mu-opioid receptor. CLSM/FCS study in live cells. PLoS ONE 3:e4008.PubMedCrossRefGoogle Scholar
  22. 22.
    Pierce KL, Premont RT, Lefkowitz RJ (2002) Seven-transmembrane receptors. Nat Rev Mol Cell Biol 3:639–650.PubMedCrossRefGoogle Scholar
  23. 23.
    Wolfe BL, Trejo J (2007) Clathrin-dependent mechanisms of G protein-coupled receptor endocytosis. Traffic 8:462–470.PubMedCrossRefGoogle Scholar
  24. 24.
    Jalink K, Moolenaar WH (2010) G protein-coupled receptors: the inside story. BioEssays 32:13–16.PubMedCrossRefGoogle Scholar
  25. 25.
    Molina PE (2006) Opioids and opiates: analgesia with cardiovascular, haemodynamic and immune implications in critical illness. J Intern Med 259:138–154.PubMedCrossRefGoogle Scholar
  26. 26.
    Tambour S, Quertemont E. (2007) Preclinical and clinical pharmacology of alcohol dependence. Fund Clin Pharmacol 21:9–28.Google Scholar
  27. 27.
    Magde D, Webb WW, Elson E (1972) Thermodynamic fluctuations in a reacting ­system - measurement by fluorescence correlation spectroscopy. Phys Rev Lett 29:705–708.CrossRefGoogle Scholar
  28. 28.
    Elson EL, Magde D (1974) Fluorescence correlation spectroscopy. Conceptual basis and theory. Biopolymers 13:1–27.Google Scholar
  29. 29.
    Ehrenberg M, Rigler R (1974) Rotational Brownian motion and fluorescence intensity fluctuations. Chem Phys 4:390–401.CrossRefGoogle Scholar
  30. 30.
    Koppel DE (1974) Statistical accuracy in fluorescence correlation spectroscopy. Phys Rev A 10:1938–1945.CrossRefGoogle Scholar
  31. 31.
    Koppel DE, Axelrod D, Schlessinger J et al (1976) Dynamics of fluorescence marker concentration as a probe of mobility. Biophys J 16:1315–1329.PubMedCrossRefGoogle Scholar
  32. 32.
    Ehrenberg M, Rigler R (1972) Polarized fluorescence and rotational Brownian motion. Chem Phys Lett 14:539–544.CrossRefGoogle Scholar
  33. 33.
    Rigler R, Mets Ü, Widengren J et al (1993) Fluorescence correlation spectroscopy with high count rate and low background. Eur Biophys J 22:169–175.CrossRefGoogle Scholar
  34. 34.
    Eigen M, Rigler R (1994) Sorting single molecules: application to diagnostics and evolutionary biotechnology. Proc Natl Acad Sci USA 91:5740–5747.PubMedCrossRefGoogle Scholar
  35. 35.
    Axelrod D, Koppel DE, Schlessinger J et al (1976) Mobility measurement by analysis of fluorescence photobleaching recovery kinetics. Biophys J 16:1055–1069.PubMedCrossRefGoogle Scholar
  36. 36.
    Elson EL, Schlessinger J, Koppel DE et al (1976) Measurement of lateral transport on cell surfaces. Prog Clin Biol Res 9:137–147.PubMedGoogle Scholar
  37. 37.
    Schlessinger J, Koppel DE, Axelrod D et al (1976) Lateral transport on cell membranes: Mobility of concanavalin A receptors on ­myoblasts. Proc Natl Acad Sci USA 73:2409–2413.PubMedCrossRefGoogle Scholar
  38. 38.
    Schlessinger J, Axelrod D, Koppel DE et al (1977) Lateral transport of a lipid probe and labeled proteins on a cell membrane. Science 195:307–309.PubMedCrossRefGoogle Scholar
  39. 39.
    Jacobson K, Elson E, Koppel D et al (1982) Fluorescence photobleaching in cell biology. Nature 295:283–284.PubMedCrossRefGoogle Scholar
  40. 40.
    Yechiel E, Edidin M (1987) Micrometer-scale domains in fibroblast plasma membranes. J Cell Biol 105:755–760.PubMedCrossRefGoogle Scholar
  41. 41.
    Berland KM, So PTC, Gratton E (1995) Two-photon fluorescence correlation spectroscopy: Method and application to the intracellular environment. Biophys J 68:694–701.PubMedCrossRefGoogle Scholar
  42. 42.
    Schwille P, Haupts U, Maiti S et al (1999) Molecular dynamics in living cells observed by fluorescence correlation spectroscopy with one- and two-photon excitation. Biophy. J 77:2251–2265.CrossRefGoogle Scholar
  43. 43.
    Denk W, Strickler JH, Webb WW (1990) Two-photon laser scanning fluorescence microscopy. Science 248: 73–76PubMedCrossRefGoogle Scholar
  44. 44.
    Zipfel WR, Williams RM, Christie A et al (2003) Live tissue intrinsic emission microscopy using multiphoton-excited native fluorescence and second harmonic generation. Proc Natl Acad Sci USA 100:7075–7080.PubMedCrossRefGoogle Scholar
  45. 45.
    Zipfel WR, Williams RM, Webb WW (2003) Nonlinear Magic: Multiphoton microscopy in the biosciences. Nature Biotech 21:1369–1377.CrossRefGoogle Scholar
  46. 46.
    Miyawaki A, Sawano A, Kogure T (2003) Lighting up cells: labelling proteins with fluorophores. Nat. Cell Biol. Suppl: S1–S7.Google Scholar
  47. 47.
    Giepmans BN, Adams SR, Ellisman MH et al (2006) The fluorescent toolbox for assessing protein location and function. Science 312:217–224.PubMedCrossRefGoogle Scholar
  48. 48.
    Marks KM, Nolan GP (2006) Chemical labeling strategies for cell biology. Nat Methods 3:591–596.PubMedCrossRefGoogle Scholar
  49. 49.
    Terasaki M, Jaffe LA (2004) Labeling of cell membranes and compartments for live cell fluorescence microscopy. Methods Cell Biol 74:469–489.PubMedCrossRefGoogle Scholar
  50. 50.
    Liu P, Ahmed S, Wohland T (2008) The F-techniques: advances in receptor protein studies. Trends Endocrinol Metab 19:181–190.PubMedCrossRefGoogle Scholar
  51. 51.
    Haustein E, Schwille P (2007) Fluorescence correlation spectroscopy: novel variations of an established technique. Annu Rev Biophys Biomol Struct 36:151–169.PubMedCrossRefGoogle Scholar
  52. 52.
    Elson EL. (2001) Fluorescence correlation spectroscopy measures molecular transport in cells. Traffic. 2:789–796.PubMedCrossRefGoogle Scholar
  53. 53.
    Thompson, N L (1991) Fluorescence correlation spectroscopy. In: Lakowicz JR (ed) Topics in Fluorescence Spectroscopy, Volume 1: Techniques. Plenum Press, New York.Google Scholar
  54. 54.
    Qian H, Elson EL (1990) On the analysis of high order moments of fluorescence fluctuations. Biophys J 57:375–380.PubMedCrossRefGoogle Scholar
  55. 55.
    Kask P, Palo K, Ullmann D et al (1999) Fluorescence-intensity distribution analysis and its application in biomolecular detection technology. Proc Natl Acad Sci USA 96:13756–13761.PubMedCrossRefGoogle Scholar
  56. 56.
    Kask P, Palo K, Fay N et al (2000) Two-dimensional fluorescence intensity distribution analysis: theory and applications. Biophys J 78:1703–1713.PubMedCrossRefGoogle Scholar
  57. 57.
    Chen Y, Müller JD, So PTC et al (1999) The photon counting histogram in fluorescence fluctuation spectroscopy. Biophys J 77:553–567.PubMedCrossRefGoogle Scholar
  58. 58.
    Hillesheim LN, Müller JD (2003) The photon counting histogram in fluorescence fluctuation spectroscopy with non-ideal photodetectors. Biophys J 85:1948–1958.PubMedCrossRefGoogle Scholar
  59. 59.
    Müller JD (2004) Cumulant analysis in fluorescence fluctuation spectroscopy. Biophys J 86:3981–3992.PubMedCrossRefGoogle Scholar
  60. 60.
    Weisshart K, Jüngel V, Briddon SJ (2004) The LSM 510 META-ConfoCor 2 system: an integrated imaging and spectroscopic platform for single-molecule detection. Curr Pharm Biotechnol 5:135–154.PubMedCrossRefGoogle Scholar
  61. 61.
    Meseth U, Wohland T, Rigler R et al (1999) Resolution of fluorescence correlation measurements. Biophys J 76:1619–1631.PubMedCrossRefGoogle Scholar
  62. 62.
    Chen Y, Wei LN, Müller JD (2003) Probing protein oligomerization in living cells with fluorescence fluctuation spectroscopy. Proc Natl Acad Sci USA 100:15492–15497.PubMedCrossRefGoogle Scholar
  63. 63.
    Petrásek Z, Hoege C, Mashaghi A et al (2008) Characterization of protein dynamics in asymmetric cell division by scanning fluorescence correlation spectroscopy. Biophys J 95:5476–5486.PubMedCrossRefGoogle Scholar
  64. 64.
    Blancquaert Y, Gao J, Derouard J et al (2008) Spatial fluorescence cross-correlation spectroscopy by means of a spatial light modulator. J Biophotonics 1:408–418.PubMedCrossRefGoogle Scholar
  65. 65.
    Eigen M, Rigler R (1994) Sorting single molecules: application to diagnostics and evolutionary biotechnology. Proc Natl Acad Sci USA 91:5740–5747.PubMedCrossRefGoogle Scholar
  66. 66.
    Rigler R, Mets Ü, Widengren J et al (1993) Fluorescence correlation spectroscopy with high count rate and low background: analysis of translational diffusion. Eur Biophys J 22:169–175.CrossRefGoogle Scholar
  67. 67.
    Koppel DE, Morgan F, Cowan AE et al (1994) Scanning concentration correlation spectroscopy using the confocal laser microscope. Biophys J 66:502–507.PubMedCrossRefGoogle Scholar
  68. 68.
    Donnert G, Eggeling C, Hell SW (2007) Major signal increase in fluorescence microscopy through dark-state relaxation. Nat Methods 4:81–86.PubMedCrossRefGoogle Scholar
  69. 69.
    Herz A (1997) Endogenous opioid systems and alcohol addiction. Psychopharmacology (Berl) 129:99–111.CrossRefGoogle Scholar
  70. 70.
    Herz A (1998) Opioid reward mechanisms: a key role in drug abuse? Can J Physiol Pharmacol 76:252–258.PubMedCrossRefGoogle Scholar
  71. 71.
    Koenig HN, Olive M (2002) Ethanol consumption patterns and conditioned place preference in mice lacking preproenkephalin. Neurosci Lett 325:75–78.PubMedCrossRefGoogle Scholar
  72. 72.
    Griesel JE, Mogil JS, Grahame NJ et al (1999) Ethanol oral self-administration is increased in mutant mice with decreased beta -endorphin expression. Brain Res 835:62–67.CrossRefGoogle Scholar
  73. 73.
    Grahame NJ, Low MJ, Cunningham CL (1998) Intravenous self-administration of ethanol in beta-endorphin-deficient mice. Alcohol Clin Exp Res 22:1093–1098.PubMedCrossRefGoogle Scholar
  74. 74.
    Roberts AJ, McDonald JS, Heyser CJ et al (2000) Mu-opioid receptor knockout mice do not self-administer alcohol. J Pharmacol Exp Ther 293:1002–1008.PubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  1. 1.Department of Clinical NeuroscienceKarolinska InstituteStockholmSweden

Personalised recommendations