University of Padua

Data related to article titled "A quantitative assay for Ca2+ uptake through normal and pathological hemichannels".

Mammano, Fabio (2022) Data related to article titled "A quantitative assay for Ca2+ uptake through normal and pathological hemichannels". [Data Collection]

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Hemichannels (HCs) are large pore hexameric structures composed of connexin (Cx) subunits that allow the exchange of ions, metabolites, and a variety of other molecules between 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 fluo-rometric measurements of Ca2+ uptake with a Ca2+-selective genetically encoded indicator (GCaMP6s) that permits 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 its usefulness for the characteriza-tion of new monoclonal antibodies (mAb) targeting the extracellular domain of the HCs. Although we developed the assay on a spinning disk confocal fluorescence microscope, the same method-ology 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.

DOI: 10.25430/
Keywords: Connexins; genetically encoded calcium indicators; monoclonal antibodies; drug discovery; ge-nodermatoses; cancer; lentivirus; bicistronic vectors.
Subjects: Life Sciences > Molecular Biology, Biochemistry, Structural Biology and Molecular Biophysics: Molecular synthesis, modification, mechanisms and interactions; biochemistry; structural biology, molecular biophysics; metabolism; signalling pathways > Macromolecular complexes including interactions involving nucleic acids, proteins, lipids and carbohydrates
Department: Departments > Dipartimento di Fisica e Astronomia "Galileo Galilei" (DFA)
Depositing User: Fabio Mammano
Date Deposited: 27 May 2022 08:19
Last Modified: 27 May 2022 08:19
Type of data: Text
Research funder: University of Padova
Research project title: Connexins as therapeutic targets addressable by human recombinant antibodies
Grant number: Prot. BIRD187130
Collection period:
1 August 201831 December 2021
Temporal coverage:
Geographic coverage: Italy, Lazio, CNR Monterotondo
Data collection method: qPCR Fluorescence microscopy Confocal microscopy
Statement on legal, ethical and access issues: No legal, ethical issues encountered in generating the data.
Data processing and preparation activities: As described in the accompanying article.
Resource language: English
Metadata language: English
Publisher: Research Data Unipd
Date: 18 May 2022
Copyright holders: The Author

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