Abstract
Chloride and protons perform important closely related roles in many cellular responses. Here we developed a ratiometric biosensor, ClopHensor, based on a highly chloride-sensitive Aequorea victoria GFP variant that is suited for the combined real-time optical detection of pH changes and chloride fluxes in live cells. We detected high chloride concentration in large dense-core exocytosis granules by targeting ClopHensor to these intracellular compartments.
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References
Faundez, V. & Hartzell, H.C. Sci. STKE 2004, re8 (2004).
Bregestovski, P., Waseem, T. & Mukhtarov, M. Front. Mol. Neurosci. 2, 15 (2009).
Planells-Cases, R. & Jentsch, T.J. Biochim Biophys Acta 1792, 173–189 (2009).
Illsley, N.P. & Verkman, A.S. Biochemistry 26, 1215–1219 (1987).
Jayaraman, S., Haggie, P., Wachter, R.M., Remington, S.J. & Verkman, A.S. J. Biol. Chem. 275, 6047–6050 (2000).
Kuner, T. & Augustine, G.J. Neuron 27, 447–459 (2000).
Galietta, L.J., Haggie, P.M. & Verkman, A.S. FEBS Lett. 499, 220–224 (2001).
Markova, O., Mukhtarov, M., Real, E., Jacob, Y. & Bregestovski, P. J. Neurosci. Methods 170, 67–76 (2008).
Wachter, R.M., Yarbrough, D., Kallio, K. & Remington, S.J. J. Mol. Biol. 301, 157–171 (2000).
Tsutsui, H., Karasawa, S., Okamura, Y. & Miyawaki, A. Nat. Methods 5, 683–685 (2008).
Jose, M., Nair, D.K., Reissner, C., Hartig, R. & Zuschratter, W. Biophys. J. 92, 2237–2254 (2007).
Arosio, D. et al. Biophys. J. 93, 232–244 (2007).
Bizzarri, R. et al. Biophys. J. 90, 3300–3314 (2006).
Wu, M.M. et al. Chem. Biol. 7, 197–209 (2000).
El Meskini, R. et al. Endocrinology 142, 864–873 (2001).
Grynkiewicz, G., Poenie, M. & Tsien, R.Y. J. Biol. Chem. 260, 3440–3450 (1985).
Valeur, B. Molecular Fluorescence: Principles and Applications (Wiley-VCH, 2002).
Zucker, R.M. & Price, O. Cytometry 44, 273–294 (2001).
Zucker, R.M. & Price, O.T. Cytometry 44, 295–308 (2001).
Zacharias, D.A., Violin, J.D., Newton, A.C. & Tsien, R.Y. Science 296, 913–916 (2002).
Wong, G., Sei, Y. & Skolnick, P. Mol. Pharmacol. 42, 996–1003 (1992).
Acknowledgements
We thank P. Faraci for his assistance in preparing NPY-ClopHensor PC12 cell samples, S. Luin for his assistance with microscope analysis, S. Sulis-Sato and G. Lanza for their help with experimental setup, A. Cereseto for critical reading of the manuscript and G. Ratto for helpful discussions. The study was supported by Fondazione Monte dei Paschi di Siena (Monte dei Paschi Foundation) and by the Italian Ministry for University and Research (Fondo per gli Investimenti della Ricerca di Base RBLA03ER38).
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D.A. conceived the sensor, designed and performed experiments, analyzed data and wrote the paper; F.R. and L.M. generated ClopHensor constructs, and designed and performed experiments; R.G. performed sensor calibrations and kinetic analysis; L.A. performed experiments and analyzed data; F.B. interpreted the data, edited the paper and gave conceptual advice.
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Arosio, D., Ricci, F., Marchetti, L. et al. Simultaneous intracellular chloride and pH measurements using a GFP-based sensor. Nat Methods 7, 516–518 (2010). https://doi.org/10.1038/nmeth.1471
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DOI: https://doi.org/10.1038/nmeth.1471
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