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Unitary permeability of gap junction channels to second messengers measured by FRET microscopy

Nature Methods volume 4, pages 353–358 (2007)Cite this article

Abstract

Gap junction channels assembled from connexin protein subunits mediate intercellular transfer of ions and metabolites. Impaired channel function is implicated in several hereditary human diseases. In particular, defective permeation of cAMP or inositol-1,4,5-trisphosphate (InsP3) through connexin channels is associated with peripheral neuropathies and deafness, respectively. Here we present a method to estimate the permeability of single gap junction channels to second messengers. Using HeLa cells that overexpressed wild-type human connexin 26 (HCx26wt) as a model system, we combined measurements of junctional conductance and fluorescence resonance energy transfer (FRET) emission ratio of biosensors selective for cAMP and InsP3. The unitary permeabilities to cAMP (47 × 10−3 ± 15 × 10−3 μm3/s) and InsP3 (60 × 10−3 ± 12 × 10−3 μm3/s) were similar, but substantially larger than the unitary permeability to lucifer yellow (LY; 7 ± 3 × 10−3 μm3/s), an exogenous tracer. This method permits quantification of defects of metabolic coupling and can be used to investigate interdependence of intercellular diffusion and cross-talk between diverse signaling pathways.

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Figure 1: HeLa cell transfection and cell volume estimate.
Figure 2: Estimate of HCx26wt permeability to LY in pairs of transfected HeLa cells.
Figure 3: Estimate of HCx26wt permeability to cAMP in pairs of transfected HeLa cells.
Figure 4: Estimate of HCx26wt permeability to InsP3 in pairs of transfected HeLa cells.

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References

  1. Sosinsky, G.E. & Nicholson, B.J. Structural organization of gap junction channels. Biochim. Biophys. Acta 1711, 99–125 (2005).

    Article  CAS  Google Scholar 

  2. Gerido, D.A. & White, T.W. Connexin disorders of the ear, skin, and lens. Biochim. Biophys. Acta 1662, 159–170 (2004).

    Article  CAS  Google Scholar 

  3. Goldberg, G.S., Valiunas, V. & Brink, P.R. Selective permeability of gap junction channels. Biochim. Biophys. Acta 1662, 96–101 (2004).

    Article  CAS  Google Scholar 

  4. Irvine, R.F. 20 years of Ins(1,4,5)P3, and 40 years before. Nat. Rev. Mol. Cell Biol. 4, 586–590 (2003).

    Article  CAS  Google Scholar 

  5. Allbritton, N.L., Meyer, T. & Stryer, L. Range of messenger action of calcium ion and inositol 1,4,5-trisphosphate. Science 258, 1812–1815 (1992).

    Article  CAS  Google Scholar 

  6. Zaccolo, M., Filippin, L., Magalhaes, P. & Pozzan, T. Heterogeneity of second messenger levels in living cells. Novartis Found. Symp. 239, 85–95, 150–159 (2001).

    CAS  PubMed  Google Scholar 

  7. Bos, J.L. Epac: a new cAMP target and new avenues in cAMP research. Nat. Rev. Mol. Cell Biol. 4, 733–738 (2003).

    Article  CAS  Google Scholar 

  8. Bacskai, B.J. et al. Spatially resolved dynamics of cAMP and protein kinase A subunits in Aplysia sensory neurons. Science 260, 222–226 (1993).

    Article  CAS  Google Scholar 

  9. Dakin, K., Zhao, Y. & Li, W.H. LAMP, a new imaging assay of gap junctional communication unveils that Ca2+ influx inhibits cell coupling. Nat. Methods 2, 55–62 (2005).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  11. Oh, S. et al. Changes in permeability caused by connexin 32 mutations underlie X-linked Charcot-Marie-Tooth disease. Neuron 19, 927–938 (1997).

    Article  CAS  Google Scholar 

  12. Qu, Y. & Dahl, G. Function of the voltage gate of gap junction channels: selective exclusion of molecules. Proc. Natl. Acad. Sci. USA 99, 697–702 (2002).

    Article  CAS  Google Scholar 

  13. Bedner, P. et al. Selective permeability of different connexin channels to the second messenger cyclic AMP. J. Biol. Chem. 281, 6673–6681 (2006).

    Article  CAS  Google Scholar 

  14. Saez, J.C., Connor, J.A., Spray, D.C. & Bennett, M.V. Hepatocyte gap junctions are permeable to the second messenger, inositol 1,4,5-trisphosphate, and to calcium ions. Proc. Natl. Acad. Sci. USA 86, 2708–2712 (1989).

    Article  CAS  Google Scholar 

  15. Beltramello, M., Piazza, V., Bukauskas, F.F., Pozzan, T. & Mammano, F. Impaired permeability to Ins(1,4,5)P3 in a mutant connexin underlies recessive hereditary deafness. Nat. Cell Biol. 7, 63–69 (2005).

    Article  CAS  Google Scholar 

  16. Ponsioen, B. et al. Detecting cAMP-induced Epac activation by fluorescence resonance energy transfer: Epac as a novel cAMP indicator. EMBO Rep. 5, 1176–1180 (2004).

    Article  CAS  Google Scholar 

  17. Tanimura, A., Nezu, A., Morita, T., Turner, R.J. & Tojyo, Y. Fluorescent biosensor for quantitative real-time measurements of inositol 1,4,5-trisphosphate in single living cells. J. Biol. Chem. 279, 38095–38098 (2004).

    Article  CAS  Google Scholar 

  18. Bastianello, S., Ciubotaru, C.D., Beltramello, M. & Mammano, F. in Three-Dimensional and Multidimensional Microscopy: Image Acquisition and Processing XI (ed. Conchello, J.-A.) Vol. 5324, 265–274 (San Jose, California, USA, 2004).

    Book  Google Scholar 

  19. Eckert, R. Gap-junctional single-channel permeability for fluorescent tracers in mammalian cell cultures. Biophys. J. 91, 565–579 (2006).

    Article  CAS  Google Scholar 

  20. 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 

  21. Brink, P.R. & Ramanan, S.V. A model for the diffusion of fluorescent probes in the septate giant axon of earthworm: axoplasmic diffusion and junctional membrane permeability. Biophys. J. 48, 299–309 (1985).

    Article  CAS  Google Scholar 

  22. Patel, S., Joseph, S.K. & Thomas, A.P. Molecular properties of inositol 1,4,5-trisphosphate receptors. Cell Calcium 25, 247–264 (1999).

    Article  CAS  Google Scholar 

  23. Peracchia, C. Chemical gating of gap junction channels; roles of calcium, pH and calmodulin. Biochim. Biophys. Acta 1662, 61–80 (2004).

    Article  CAS  Google Scholar 

  24. Paemeleire, K. et al. Intercellular calcium waves in HeLa cells expressing GFP-labeled connexin 43, 32, or 26. Mol. Biol. Cell 11, 1815–1827 (2000).

    Article  CAS  Google Scholar 

  25. Bicego, M. et al. Pathogenetic role of the deafness-related M34T mutation of Cx26. Hum. Mol. Genet. 15, 2569–2587 (2006).

    Article  CAS  Google Scholar 

  26. Matsu-ura, T. et al. Cytosolic inositol 1,4,5-trisphosphate dynamics during intracellular calcium oscillations in living cells. J. Cell Biol. 173, 755–765 (2006).

    Article  CAS  Google Scholar 

  27. Nikolaev, V.O., Gambaryan, S. & Lohse, M.J. Fluorescent sensors for rapid monitoring of intracellular cGMP. Nat. Methods 3, 23–25 (2006).

    Article  CAS  Google Scholar 

  28. Beltramello, M. et al. Permeability and gating properties of human connexins 26 and 30 expressed in HeLa cells. Biochem. Biophys. Res. Commun. 305, 1024–1033 (2003).

    Article  CAS  Google Scholar 

  29. Downes, C.P., Mussat, M.C. & Michell, R.H. The inositol trisphosphate phosphomonoesterase of the human erythrocyte membrane. Biochem. J. 203, 169–177 (1982).

    Article  CAS  Google Scholar 

  30. Mammano, F. et al. An optical recording system based on a fast CCD sensor for biological imaging. Cell Calcium 25, 115–123 (1999).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

This work was funded by grants from Telethon Italy (GGP05131) and the European commission FP6 Integrated Project EuroHear (LSHG-CT-20054-512063) under the Sixth Research Frame Program of The European Union (to F.M.) and from Fondazione CARIPARO (to S.P.). M.Z. is supported by Telethon Italy (TCP00089, GGP05113), the Italian Cystic Fibrosis Research Foundation, the Fondazione Compagnia di San Paolo and the HFSPO (RGP1/2005). We thank K. Willecke (University of Bonn), R. Bruzzone (Institute Pasteur), K. Jalink (The Netherlands Cancer Institute) and A. Tanimura (Health Sciences University of Hokkaido) for the gifts of HeLa cells, HCx26wt, H30 and LIBRA, respectively, and T. Pozzan (University of Padova) for helpful discussions and constructive criticism.

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Author notes

  1. Victor H Hernandez and Mario Bortolozzi: These authors contributed equally to this work.

Authors and Affiliations

  1. Istituto Veneto di Medicina Molecolare, Fondazione per la Ricerca Biomedica Avanzata, Padova, 35129, Italy

    Victor H Hernandez, Mario Bortolozzi, Vanessa Pertegato, Martina Beltramello, Manuela Zaccolo, Sergio Pantano & Fabio Mammano

  2. Istituto Dulbecco Telethon, Fondazione per la Ricerca Biomedica Avanzata, Padova, 35129, Italy

    Vanessa Pertegato & Manuela Zaccolo

  3. Dipartimento di Fisica 'G. Galilei' dell'Università di Padova, Padova, 35131, Italy

    Michele Giarin & Fabio Mammano

  4. Consorzio Nazionale Interuniversitario per le Scienze Fisiche della Materia, Padova, 35131, Italy

    Sergio Pantano & Fabio Mammano

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Correspondence to Fabio Mammano.

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Supplementary information

Supplementary Fig. 1

Structural models of the HCx26wt connexon and permeant molecules. (PDF 1596 kb)

Supplementary Methods (DOC 76 kb)

Supplementary Discussion (DOC 60 kb)

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Hernandez, V., Bortolozzi, M., Pertegato, V. et al. Unitary permeability of gap junction channels to second messengers measured by FRET microscopy. Nat Methods 4, 353–358 (2007). https://doi.org/10.1038/nmeth1031

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