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| A Quantitative Assay for Ca2+ Uptake through Normal and Pathological Hemichannels | |
| 2022-07 | |
| Source Publication | INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES
 |
| EISSN | 1422-0067 |
| Volume | 23Issue:13 |
| Status | 已发表 |
| DOI | 10.3390/ijms23137337 |
| 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. |
| Keyword | connexins genetically encoded calcium indicators monoclonal antibodies drug discovery genodermatoses cancer lentivirus bicistronic vectors |
| URL | 查看原文 |
| Indexed By | SCI ; SCIE |
| Language | 英语 |
| Funding Project | University of Padova[BIRD187130] ; Fondazione Telethon[GGP19148] ; Italian Ministry of Education, Universities and Research (MUR)["PRIN 20175C22WM","PRIN 2017FTJ5ZE"] |
| WOS Research Area | Biochemistry & Molecular Biology ; Chemistry |
| WOS Subject | Biochemistry & Molecular Biology ; Chemistry, Multidisciplinary |
| WOS ID | WOS:000823482000001 |
| Publisher | MDPI |
| Citation statistics | |
| Document Type | 期刊论文 |
| Identifier | https://kms.shanghaitech.edu.cn/handle/2MSLDSTB/214799 |
| Collection | 免疫化学研究所_特聘教授组_Michael Levitt组 免疫化学研究所_特聘教授组_功能筛选实验室 |
| Corresponding Author | Sidhu, Sachdev S.; Mammano, Fabio |
| Affiliation | 1.CNR Inst Biochem & Cell Biol, I-00015 Rome, Italy 2.ShanghaiTech Univ, Shanghai Inst Adv Immunochem Studies, Shanghai 201210, Peoples R China 3.Univ Toronto, Donnelly Ctr, Toronto, ON M5S 3E1, Canada 4.Univ Padua, Dept Phys & Astron G Galilei, I-35131 Padua, Italy 5.Ist Italiano Tecnol, Opt Approaches Brain Funct Lab, I-16163 Genoa, Italy |
| Recommended Citation GB/T 7714 | Nardin, Chiara,Tettey-Matey, Abraham,Donati, Viola,et al. A Quantitative Assay for Ca2+ Uptake through Normal and Pathological Hemichannels[J]. INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES,2022,23(13). |
| APA | Nardin, Chiara.,Tettey-Matey, Abraham.,Donati, Viola.,Marazziti, Daniela.,Di Pietro, Chiara.,...&Mammano, Fabio.(2022).A Quantitative Assay for Ca2+ Uptake through Normal and Pathological Hemichannels.INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES,23(13). |
| MLA | Nardin, Chiara,et al."A Quantitative Assay for Ca2+ Uptake through Normal and Pathological Hemichannels".INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES 23.13(2022). |
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