A Quantitative Assay for Ca2+ Uptake through Normal and Pathological Hemichannels |
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| Authors: | Chiara Nardin Abraham Tettey-Matey Viola Donati Daniela Marazziti Chiara Di Pietro Chiara Peres Marcello Raspa Francesco Zonta Guang Yang Maryna Gorelik Serena Singh Lia Cardarelli Sachdev S. Sidhu Fabio Mammano |
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| Affiliation: | 1.CNR Institute of Biochemistry and Cell Biology, Monterotondo, 00015 Rome, Italy; (C.N.); (A.T.-M.); (V.D.); (D.M.); (C.D.P.); (C.P.); (M.R.);2.Shanghai Institute for Advanced Immunochemical Studies, ShanghaiTech University, Shanghai 201210, China; (F.Z.); (G.Y.);3.The Donnelly Centre, University of Toronto, Toronto, ON M5S 3E1, Canada; (M.G.); (S.S.); (L.C.);4.Department of Physics and Astronomy “G. Galilei”, University of Padua, 35131 Padua, Italy |
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| Abstract: | Connexin (Cx) hemichannels (HCs) are large pore hexameric structures that allow the exchange of ions, metabolites and a variety of other molecules between the cell cytoplasm and extracellular milieu. HC inhibitors are attracting growing interest as drug candidates because deregulated fluxes through HCs have been implicated in a plethora of genetic conditions and other diseases. HC activity has been mainly investigated by electrophysiological methods and/or using HC-permeable dye uptake measurements. Here, we present an all-optical assay based on fluorometric measurements of ionized calcium (Ca2+) uptake with a Ca2+-selective genetically encoded indicator (GCaMP6s) that permits the optical tracking of cytosolic Ca2+ concentration ([Ca2+]cyt) changes with high sensitivity. We exemplify use of the assay in stable pools of HaCaT cells overexpressing human Cx26, Cx46, or the pathological mutant Cx26G45E, under control of a tetracycline (Tet) responsive element (TRE) promoter (Tet-on). We demonstrate the usefulness of the assay for the characterization of new monoclonal antibodies (mAbs) targeting the extracellular domain of the HCs. Although we developed the assay on a spinning disk confocal fluorescence microscope, the same methodology can be extended seamlessly to high-throughput high-content platforms to screen other kinds of inhibitors and/or to probe HCs expressed in primary cells and microtissues. |
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| Keywords: | connexins genetically encoded calcium indicators monoclonal antibodies drug discovery genodermatoses cancer lentivirus bicistronic vectors |
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