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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Data related to article titled "A quantitative assay for Ca2+ uptake through normal and pathological hemichannels". (deposited 27 May 2022 08:19)
- Data related to article titled "A quantitative assay for Ca2+ uptake through normal and pathological hemichannels". (deposited 09 Jan 2023 10:02) [Currently Displayed]
Collection description
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/researchdata.cab.unipd.it.00000645 |
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| 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 Life Sciences > Molecular Biology, Biochemistry, Structural Biology and Molecular Biophysics: Molecular synthesis, modification, mechanisms and interactions; biochemistry; structural biology, molecular biophysics; metabolism; signalling pathways > Molecular biophysics (e.g. single-molecule approaches, bioenergetics, fluorescence) Life Sciences > Applied Medical Technologies, Diagnostics, Therapies, and Public Health: Development of treatment of diseases, pharmacology, clinical medicine, regenerative medicine, epidemiology and public for diagnosis, monitoring and tools health > Pharmacology and pharmacogenomics (including drug discovery and design, drug delivery and therapy, toxicology) |
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| Department: | Departments > Dipartimento di Fisica e Astronomia "Galileo Galilei" (DFA) | ||||||
| Depositing User: | Fabio Mammano | ||||||
| Date Deposited: | 09 Jan 2023 10:02 | ||||||
| Last Modified: | 24 Jan 2023 08:27 | ||||||
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| Type of data: | Text | ||||||
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| Research funder: | University of Padova | ||||||
| Research project title: | Connexins as therapeutic targets addressable by human recombinant antibodies | ||||||
| Grant number: | Prot. BIRD187130 | ||||||
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| 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 | ||||||
| Additional information: | PMID: 35806342; PMCID: PMC9266989; | ||||||
| Publisher: | Research Data Unipd | ||||||
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| Date: | 18 May 2022 | ||||||
| Copyright holders: | The Author | ||||||
| URI: | https://researchdata.cab.unipd.it/id/eprint/653 |
orcid.org/0000-0003-3751-1691
