A Quantitative Assay for Ca2+ Uptake through Normal and Pathological Hemichannels

doi:10.3390/ijms23137337

	A Quantitative Assay for Ca2+ Uptake through Normal and Pathological Hemichannels
	Nardin, Chiara 1,5; Tettey-Matey, Abraham 1; Donati, Viola 1; Marazziti, Daniela 1; Di Pietro, Chiara 1; Peres, Chiara 1; Raspa, Marcello 1; Zonta, Francesco2 ; Yang, Guang2 ; Gorelik, Maryna 3; Singh, Serena 3; Cardarelli, Lia 3; Sidhu, Sachdev S.3; Mammano, Fabio 1,4
	2022-07
发表期刊	INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES
EISSN	1422-0067
卷号	23 期号:13
发表状态	已发表
DOI	10.3390/ijms23137337
摘要	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.
关键词	connexins genetically encoded calcium indicators monoclonal antibodies drug discovery genodermatoses cancer lentivirus bicistronic vectors
URL	查看原文
收录类别	SCI ; SCIE
语种	英语
资助项目	University of Padova[BIRD187130] ; Fondazione Telethon[GGP19148] ; Italian Ministry of Education, Universities and Research (MUR)["PRIN 20175C22WM","PRIN 2017FTJ5ZE"]
WOS研究方向	Biochemistry & Molecular Biology ; Chemistry
WOS类目	Biochemistry & Molecular Biology ; Chemistry, Multidisciplinary
WOS记录号	WOS:000823482000001
出版者	MDPI
引用统计
文献类型	期刊论文
条目标识符	https://kms.shanghaitech.edu.cn/handle/2MSLDSTB/214799
专题	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
通讯作者	Sidhu, Sachdev S.; Mammano, Fabio
作者单位	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
推荐引用方式 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).