The Use of FRET Microscopy to Elucidate Steady State Channel Conformational Rearrangements and G Protein Interaction with the GIRK Channels

  • Adi Raveh
  • Inbal Riven
  • Eitan Reuveny
Part of the Methods in Molecular Biology book series (MIMB, volume 491)


while X-ray crystallography provides extremely high-resolution snapshot of protein structure, it lacks the ability to provide dynamic information on the processes involving conformational rearrangements of the protein. Methods to record protein conformational dynamics are present, in particular those that are based on fluorescence measurements, and are now more and more utilized in studying proteins in their natural environment. Here we describe the use of fluorescence resonance energy transfer (FRET) technique to monitor the conformational rearrangements associated with the gating of the G protein-coupled potassium channel (GIRK/Kir3.x), and its relation with the G protein subunits. The FRET technique is combined with total internal fluorescence (TIRF) microscopy, and allows the dissection of the signal originating from channel proteins that reside exclusively in the plasma membrane. Since most of the components associated with GIRK channel gating are intracellular, that involve various biochemical steps, proteins were labeled with genetically encoded variants of the green fluorescence protein and signals were acquired from live cells in culture. Using these methodologies we were able to show that gating conformational rearrangements, i.e. the opening of the channel, involve the rotation and expansion of the channel subunits cytosolic termini, along the channel's central axis. In addition, the G proteins that trigger this process reside very close to the channel, to ensure high signaling specificity and to provide temporal precision of the gating process.

Key words

TIRF microcopy FRET GIRK GPCR Fluorescence spectroscopy Anisotropy measurements. 


  1. 1.
    Lakowicz, J. R. (2006) Principles of fluorescence spectroscopy, 3rd Edition. Springer, Berlin.CrossRefGoogle Scholar
  2. 2.
    Stryer, L. (1978) Fluorescence energy transfer as a spectroscopic ruler. Annu. Rev. Bio-chem. 47, 819–846.Google Scholar
  3. 3.
    Wallrabe, H. and Periasamy, A. (2005) Imaging protein molecules using FRET and FLIM microscopy. Curr. Opin. Biotech-nol. 16, 19–27.CrossRefGoogle Scholar
  4. 4.
    Nguyen, A. W. and Daugherty, P. S. (2005) Evolutionary optimization of fluorescent proteins for intracellular FRET. Nat. Bio-technol. 23, 355–360.CrossRefGoogle Scholar
  5. 5.
    Jares-Erijman, E. A. and Jovin, T. M. (2003) FRET imaging. Nat. Biotechnol. 21, 1387–1395.CrossRefPubMedGoogle Scholar
  6. 6.
    Miyawaki, A. and Tsien, R. Y. (2000) Monitoring protein conformations and interactions by fluorescence resonance energy transfer between mutants of green fluorescent protein. Methods Enzymol. 327, 472–500.CrossRefPubMedGoogle Scholar
  7. 7.
    Bastiaens, P. I., Majoul, I. V., Verveer, P. J., Soling, H. D., and Jovin, T. M. (1996) Imaging the intracellular trafficking and state of the AB5 quaternary structure of cholera toxin. EMBO J. 15, 4246–4253.PubMedGoogle Scholar
  8. 8.
    Tsien, R. Y. (1998) The green fluorescent protein. Annu. Rev. Biochem. 67, 509–544.CrossRefPubMedGoogle Scholar
  9. 9.
    Riven, I., Iwanir, S., and Reuveny, E. (2006) GIRK channel activation involves a local rearrangement of a preformed G protein channel complex. Neuron 51, 561–573.CrossRefPubMedGoogle Scholar
  10. 10.
    Riven, I., Kalmanzon, E., Segev, L., and Reuveny, E. (2003) Conformational rearrangements associated with the gating of the G protein-coupled potassium channel revealed by FRET microscopy. Neuron 38, 225–235.CrossRefPubMedGoogle Scholar
  11. 11.
    Luscher, C., Jan, L. Y., Stoffel, M., Malenka, R. C., and Nicoll, R. A. (1997) G protein-coupled inwardly rectifying K+ channels (GIRKs) mediate postsynaptic but not presynaptic transmitter actions in hip-pocampal neurons. Neuron 19, 687–695.CrossRefPubMedGoogle Scholar
  12. 12.
    Kuo, A., Gulbis, J. M., Antcliff, J. F., Rahman, T., Lowe, E. D., Zimmer, J., Cuthbertson, J., Ashcroft, F. M., Ezaki, T., and Doyle, D. A. (2003) Crystal structure of the potassium channel KirBac1.1 in the closed state. Science 300, 1922–1926.CrossRefPubMedGoogle Scholar
  13. 13.
    Nishida, M. and MacKinnon, R. (2002) Structural basis of inward rectification, cytoplasmic pore of the G protein-gated inward rectifier GIRK1 at 1.8 A resolution. Cell 111, 957–965.CrossRefPubMedGoogle Scholar
  14. 14.
    Pegan, S., Arrabit, C., Zhou, W., Kwiatkowski, W., Collins, A., Slesinger, P. A., and Choe, S. (2005) Cytoplasmic domain structures of Kir2.1 and Kir3.1 show sites for modulating gating and rectification. Nat. Neuro-sci. 8, 279–287.CrossRefGoogle Scholar
  15. 15.
    Doyle, D. A., Cabral, J. M., Pfuetzner, R. A., Kuo, A., Gulbis, J. M., Cohen, S. L., Chait, B. T., and MacKinnon, R. (1998) The structure of the potassium channel, molecular basis of K+ conduction and selectivity. Science 280, 69–77.CrossRefPubMedGoogle Scholar
  16. 16.
    Jiang, Y., Lee, A., Chen, J., Cadene, M., Chait, B. T., and MacKinnon, R. (2002) Crystal structure and mechanism of a calcium-gated potassium channel. Nature 417, 515–522.CrossRefPubMedGoogle Scholar
  17. 17.
    Nishida, M., Cadene, M., Chait, B. T., and MacKinnon, R. (2007) Crystal structure of a Kir3.1-prokaryotic Kir channel chimera. EMBO J. 26, 4005–4015.CrossRefPubMedGoogle Scholar
  18. 18.
    Bichet, D., Haass, F. A., and Jan, L. Y. (2003) Merging functional studies with structures of inward-rectifier K+ channels. Nat. Rev. Neurosci. 4, 957–967.CrossRefPubMedGoogle Scholar
  19. 19.
    Tucker, S. J., Pessia, M., and Adelman, J. P. (1996) Muscarine-gated K+ channel, subu-nit stoichiometry and structural domains essential for G protein stimulation. Am. J. Physiol. 271, H379–H385.PubMedGoogle Scholar
  20. 20.
    Silverman, S. K., Lester, H. A., and Dougherty, D. A. (1996) Subunit stoichiometry of a heteromultimeric G protein-coupled inward-rectifier K+ channel. J. Biol. Chem. 271, 30524–30528.CrossRefPubMedGoogle Scholar
  21. 21.
    Sadja, R., Alagem, N., and Reuveny, E. (2002) Graded contribution of the Gbeta gamma binding domains to GIRK channel activation. Proc. Natl Acad. Sci. USA 99, 10783–10788.CrossRefPubMedGoogle Scholar
  22. 22.
    Heim, R. and Tsien, R. Y. (1996) Engineering green fluorescent protein for improved brightness, longer wavelengths and fluorescence resonance energy transfer. Curr. Biol. 6, 178–182.CrossRefPubMedGoogle Scholar
  23. 23.
    Axelrod, D. (1989). Total internal reflection fluorescence microscopy. Methods Cell Biol. 30, 245–270.CrossRefGoogle Scholar
  24. 24.
    Axelrod, D., Thompson, N. L., and Burghardt, T. P. (1983) Total internal inflection fluorescent microscopy. J. Microsc. 129,, 19–28.PubMedGoogle Scholar
  25. 25.
    Reuveny, E., Slesinger, P. A., Inglese, J., Morales, J. M., Iniguez-Lluhi, J. A., Lefkowitz, R. J., Bourne, H. R., Jan, Y. N., and Jan, L. Y. (1994) Activation of the cloned muscarinic potassium channel by G protein beta gamma subunits. Nature 370, 143–146CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2008

Authors and Affiliations

  • Adi Raveh
    • 1
  • Inbal Riven
    • 2
  • Eitan Reuveny
    • 1
  1. 1.Department of Biological ChemistryWeizmann Institute of ScienceRehovotIsrael
  2. 2.Department of Physiology and Pharmacology, Sackler School of MedicineTel Aviv UniversityTel AvivIsrael

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