Summary
Fluorescent dyes sensitive to changes in intracellular calcium have become increasingly popular in G protein-coupled receptor (GPCR) drug discovery for several reasons. First of all, the assays using the dyes are easy to perform and are of low cost compared to other assays. Second, most non-Gαq-coupled GPCRs can be tweaked to modulate intracellular calcium by co-transfection with promiscuous or chimeric/mutated G proteins making the calcium assays broadly applicable in GPCR research. Third, the price of instruments capable of measuring fluorescent-based calcium indicators has become significantly less making them obtainable even for academic groups. Here, we present a protocol for measuring changes in intracellular calcium levels in living mammalian cells based on the fluorescent calcium binding dye, fluo-4.
Key words
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Minta, A., Kao, J. P. Y., and Tsien, R. Y. (1989) Fluorescent indicators for cytosolic calcium based on rhodamine and fluorescein chromophores. J. Biol. Chem. 264, 8171–8178.
Gee, K. R., Brown, K. A., Chen, W. N. U., Bishop-Stewart, J., Gray, D., and Johnson, I. (2000) Chemical and physiological characterization of fluo-4 Ca2+-indicator dyes. Cell Calcium 27, 97–106.
Kostenis, E. (2006) G proteins in drug screening: From analysis of receptor-G protein specificity to manipulation of GPCR-mediated signalling pathways. Curr. Pharm. Des. 12, 1703–1715.
Conklin, B. R., Farfel, Z., Lustig, K. D., Julius, D., and Bourne, H. R. (1993) Substitution of three amino acids switches receptor specificity of Gqα to that of Giα. Nature 363, 274–276.
Offermanns, S., and Simon, M. I. (1995) G alpha 15 and G alpha 16 couple a wide variety of receptors to phospholipase C. J. Biol. Chem. 270, 15175–15180.
Heydorn, A., Ward, R. J., Jorgensen, R., Rosenkilde, M. M., Frimurer, T. M., Milligan, G., and Kostenis, E. (2004) Identification of a novel site within G protein alpha subunits important for specificity of receptor-G protein interaction Mol. Pharmacol. 66, 250–259.
Bräuner-Osborne, H., and Krogsgaard-Larsen, P. (1999) Functional pharmacology of cloned heterodimeric GABAB receptors expressed in mammalian cells. Br. J. Pharmacol. 128, 1370–1374.
Christiansen, B., Hansen, K. B., Wellendorph, P., and Bräuner-Osborne, H. (2007) Pharmacological characterization of mouse GPRC6A, an L-α-amino acid receptor with ability to sense divalent cations. Br. J. Pharmacol. 150, 798–807.
Takahashi, A., Camacho, P., Lechleiter, J. D., and Herman, B. (1999) Measurement of intracellular calcium. Physiol. Rev. 79, 1089–1125.
Kenakin, T. P., and Morgan, P. H. (1989) Theoretical effects of single and multiple transducer receptor coupling proteins on estimates of the relative potency of agonists. Mol. Pharmacol. 35, 214–222.
Lundholt, B. K., Scudder, K. M., and Pagliaro, L. (2003) A simple technique for reducing edge effect in cell-based assays. J. Biomol. Screen. 8, 566–570.
Bjerrum, E. J., Kristensen, A. S., Pickering, D. S., Greenwood, J. R., Nielsen, B., Liljefors, T., Schousboe, A., Bräuner-Osborne, H., and Madsen, U. (2003) Design, synthesis, and pharmacology of a highly subtype-selective GluR1/2 agonist, (RS)-2-amino-3-(4-chloro-3-hydroxy-5-isoxazolyl)propionic acid (Cl-HIBO). J. Med. Chem. 46, 2246–2249.
Acknowledgments
Our work on FLIPR® assays of intracellular calcium has been supported by The Danish Medical Research Council, the Lundbeck Foundation, the Villum Kann Rasmussen Foundation, the Novo Nordisk Foundation, Fonden af 17-12-1981, the Augustinus Foundation, and the Direktør Ib Henriksen Foundation.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2009 Humana Press, a part of Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Hansen, K.B., Bräuner-Osborne, H. (2009). FLIPR® Assays of Intracellular Calcium in GPCR Drug Discovery. In: Leifert, W. (eds) G Protein-Coupled Receptors in Drug Discovery. Methods in Molecular Biology, vol 552. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-60327-317-6_19
Download citation
DOI: https://doi.org/10.1007/978-1-60327-317-6_19
Published:
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-60327-316-9
Online ISBN: 978-1-60327-317-6
eBook Packages: Springer Protocols