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LAMP, a new imaging assay of gap junctional communication unveils that Ca2+ influx inhibits cell coupling

Nature Methods volume 2, pages 55–62 (2005)Cite this article

Abstract

Using a new class of photo-activatible fluorophores, we have developed a new imaging technique for measuring molecular transfer rates across gap junction connexin channels in intact living cells. This technique, named LAMP, involves local activation of a molecular fluorescent probe, NPE-HCCC2/AM, to optically label a cell. Subsequent dye transfer through gap junctions from labeled to unlabeled cells was quantified by fluorescence microscopy. Additional uncagings after prior dye transfers reached equilibrium enabled multiple measurements of dye transfer rates in the same coupled cell pair. Measurements in the same cell pair minimized variation due to differences in cell volume and number of gap junctions, allowing us to track acute changes in gap junction permeability. We applied the technique to study the regulation of gap junction coupling by intracellular Ca2+ ([Ca2+]i). Although agonist or ionomycin exposure can raise bulk [Ca2+]i to levels higher than those caused by capacitative Ca2+ influx, the LAMP assay revealed that only Ca2+ influx through the plasma membrane store-operated Ca2+ channels strongly reduced gap junction coupling. The noninvasive and quantitative nature of this imaging technique should facilitate future investigations of the dynamic regulation of gap junction communication.

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Figure 1: A new imaging assay of gap junctional communication based on a new generation of caged fluorophores.
Figure 2: A non-invasive and quantitative imaging assay of gap junctional communication.
Figure 3: Agonist or ionomycin stimulated [Ca2+]i rises did not reduce gap junction coupling.
Figure 4: Capacitative Ca2+ influx inhibits gap junction coupling.

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References

  1. De Maio, A., Vega, V.L. & Contreras, J.E. Gap junctions, homeostasis, and injury. J. Cell. Physiol. 191, 269–282 (2002).

    Article  CAS  Google Scholar 

  2. Bennett, M.V. & Zukin, R.S. Electrical coupling and neuronal synchronization in the Mammalian brain. Neuron 41, 495–511 (2004).

    Article  CAS  Google Scholar 

  3. Peracchia, C. (ed.) Gap junctions molecular basis of cell communication in health and disease (Academic Press, 2000).

    Google Scholar 

  4. Goodenough, D.A., Goliger, J.A. & Paul, D.L. Connexins, connexons and intercellular communication. Annu. Rev. Biochem. 65, 475–502 (1996).

    Article  CAS  Google Scholar 

  5. Harris, A.L. Emerging issues of connexin channels: biophysics fills the gap. Q. Rev. Biophys. 34, 325–472 (2001).

    Article  CAS  Google Scholar 

  6. Wade, M.H., Trosko, J.E. & Schindler, M. A fluorescence photobleaching assay of gap junction-mediated communication between human cells. Science 232, 525–528 (1986).

    Article  CAS  Google Scholar 

  7. Van Rijen, H.V.M., Wilders, R., Rook, M.B. & Jongsma, H.J. in Connexin methods and protocols (eds. Bruzzone, R. & Giaume, C.) 269–292 (Humana, Totowa, New Jersey, 2001).

    Google Scholar 

  8. Valiunas, V., Beyer, E.C. & Brink, P.R. Cardiac gap junction channels show quantitative differences in selectivity. Circ. Res. 91, 104–111 (2002).

    Article  CAS  Google Scholar 

  9. Wilders, R. & Jongsma, H.J. Limitations of the dual voltage clamp method in assaying conductance and kinetics of gap junction channels. Biophys. J. 63, 942–953 (1992).

    Article  CAS  Google Scholar 

  10. Zhao, Y. et al. New caged coumarin fluorophores with extraordinary uncaging cross sections suitable for biological imaging applications. J. Am. Chem. Soc. 126, 4653–4663 (2004).

    Article  CAS  Google Scholar 

  11. Davidson, J.S. & Baumgarten, I.M. Glycyrrhetinic acid derivatives: a novel class of inhibitors of gap-junctional intercellular communication. Structure-activity relationships. J. Pharmacol. Exp. Ther. 246, 1104–1107 (1988).

    CAS  Google Scholar 

  12. Peters, R. Nuclear envelope permeability measured by fluorescence microphotolysis of single liver cell nuclei. J. Biol. Chem. 258, 11427–11429 (1983).

    CAS  PubMed  Google Scholar 

  13. Biegon, R.P., Atkinson, M.M., Liu, T.F., Kam, E.Y. & Sheridan, J.D. Permeance of Novikoff hepatoma gap junctions: quantitative video analysis of dye transfer. J. Membr. Biol. 96, 225–233 (1987).

    Article  CAS  Google Scholar 

  14. Oliveira-Castro, G.M. & Loewenstein, W.R. Junctional membrane permeability: effects of divalent cations. J. Membr. Biol. 5, 51–77 (1971).

    Article  CAS  Google Scholar 

  15. Noma, A. & Tsuboi, N. Dependence of junctional conductance on proton, calcium and magnesium ions in cardiac paired cells of guinea-pig. J. Physiol. (Lond.) 382, 193–211 (1987).

    Article  CAS  Google Scholar 

  16. Lazrak, A. & Peracchia, C. Gap junction gating sensitivity to physiological internal calcium regardless of pH in Novikoff hepatoma cells. Biophys. J. 65, 2002–2012 (1993).

    Article  CAS  Google Scholar 

  17. Spray, D.C., Stern, J.H., Harris, A.L. & Bennett, M.V. Gap junctional conductance: comparison of sensitivities to H and Ca ions. Proc. Natl. Acad. Sci. USA 79, 441–445 (1982).

    Article  CAS  Google Scholar 

  18. Firek, L. & Weingart, R. Modification of gap junction conductance by divalent cations and protons in neonatal rat heart cells. J. Mol. Cell. Cardiol. 27, 1633–1643 (1995).

    Article  CAS  Google Scholar 

  19. Berridge, M.J. Inositol trisphosphate and calcium signalling. Nature 361, 315–325 (1993).

    Article  CAS  Google Scholar 

  20. Takemura, H., Hughes, A.R., Thastrup, O. & Putney, J.W., Jr. Activation of calcium entry by the tumor promoter thapsigargin in parotid acinar cells. Evidence that an intracellular calcium pool and not an inositol phosphate regulates calcium fluxes at the plasma membrane. J. Biol. Chem. 264, 12266–12271 (1989).

    CAS  Google Scholar 

  21. Grynkiewicz, G., Poenie, M. & Tsien, R.Y. A new generation of Ca2+ indicators with greatly improved fluorescence properties. J. Biol. Chem. 260, 3440–3450 (1985).

    CAS  Google Scholar 

  22. Minta, A., Kao, J.P.Y. & Tsien, R.Y. Fluorescent Indicators For Cytosolic Calcium Based On Rhodamine and Fluorescein Chromophores. J. Biol. Chem. 264, 8171–8178 (1989).

    CAS  Google Scholar 

  23. Kwan, C.Y., Takemura, H., Obie, J.F., Thastrup, O. & Putney, J.W., Jr. Effects of MeCh, thapsigargin, and La3+ on plasmalemmal and intracellular Ca2+ transport in lacrimal acinar cells. Am. J. Physiol. 258, C1006–C1015 (1990).

    Article  CAS  Google Scholar 

  24. Marsault, R., Murgia, M., Pozzan, T. & Rizzuto, R. Domains of high Ca2+ beneath the plasma membrane of living A7r5 cells. EMBO J. 16, 1575–1581 (1997).

    Article  CAS  Google Scholar 

  25. Davies, E.V. & Hallett, M.B. High micromolar Ca2+ beneath the plasma membrane in stimulated neutrophils. Biochem. Biophys. Res. Commun. 248, 679–683 (1998).

    Article  CAS  Google Scholar 

  26. Unwin, P.N. & Ennis, P.D. Two configurations of a channel-forming membrane protein. Nature 307, 609–613 (1984).

    Article  CAS  Google Scholar 

  27. Muller, D.J., Hand, G.M., Engel, A. & Sosinsky, G.E. Conformational changes in surface structures of isolated connexin 26 gap junctions. EMBO J. 21, 3598–3607 (2002).

    Article  CAS  Google Scholar 

  28. Turin, L. & Warner, A.E. Intracellular pH in early Xenopus embryos: its effect on current flow between blastomeres. J. Physiol. (Lond.) 300, 489–504 (1980).

    Article  CAS  Google Scholar 

  29. Peracchia, C., Sotkis, A., Wang, X.G., Peracchia, L.L. & Persechini, A. Calmodulin directly gates gap junction channels. J. Biol. Chem. 275, 26220–26224 (2000).

    Article  CAS  Google Scholar 

  30. Jurado, L.A., Chockalingam, P.S. & Jarrett, H.W. Apocalmodulin. Physiol. Rev. 79, 661–682 (1999).

    Article  CAS  Google Scholar 

  31. DeVries, S.H. & Schwartz, E.A. Hemi-gap-junction channels in solitary horizontal cells of the catfish retina. J. Physiol. (Lond.) 445, 201–230 (1992).

    Article  CAS  Google Scholar 

  32. Goodenough, D.A. & Paul, D.L. Beyond the gap: functions of unpaired connexon channels. Nat. Rev. Mol. Cell Biol. 4, 285–294 (2003).

    Article  CAS  Google Scholar 

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Acknowledgements

This research was supported by a research grant (I–1510) from the Welch Foundation and a Career Development Award from the American Diabetes Associations to W.-H. Li. We thank F. Grinnell for providing primary human fibroblasts. We also thank K. Luby-Phelps, R. Anderson and F. Grinnell for critical comments on the manuscript.

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  1. Departments of Cell Biology and of Biochemistry, University of Texas Southwestern Medical Center at Dallas, 5323 Harry Hines Blvd., Dallas, 75390, TX, USA

    Kenneth Dakin, YuRui Zhao & Wen-Hong Li

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  1. Kenneth Dakin
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Correspondence to Wen-Hong Li.

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Supplementary information

Supplementary Fig. 1

Primary human foreskin fibroblasts express connexin 43 and form gap junctions in culture. (PDF 745 kb)

Supplementary Fig. 2

HCCC2 diffuses rapidly intracellularly. (PDF 425 kb)

Supplementary Fig. 3

[Ca2+]i fluctuations in HF by Fura-2 imaging. (PDF 185 kb)

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Dakin, K., Zhao, Y. & Li, WH. LAMP, a new imaging assay of gap junctional communication unveils that Ca2+ influx inhibits cell coupling. Nat Methods 2, 55–62 (2005). https://doi.org/10.1038/nmeth730

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