Mammano, Fabio
(2022)
Data related to article titled "A quantitative assay for Ca2+ uptake through normal and pathological hemichannels".
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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)
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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 |
| 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 |
| Creators/Authors: |
|
| Type of data: |
Text |
| Contributors: |
| Contribution | Name | Email |
|---|
| Author | Mammano, Fabio | fabio.mammano@unipd.it |
|
| Research funder: |
University of Padova |
| Research project title: |
Connexins as therapeutic targets addressable by human recombinant antibodies |
| Grant number: |
Prot. BIRD187130 |
| Collection period: |
| From | To |
|---|
| 1 August 2018 | 31 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 |
| URI: |
https://researchdata.cab.unipd.it/id/eprint/645 |
| Creators/Authors: |
|
| Type of data: |
Text |
| Contributors: |
| Contribution | Name | Email |
|---|
| Author | Mammano, Fabio | fabio.mammano@unipd.it |
|
| Research funder: |
University of Padova |
| Research project title: |
Connexins as therapeutic targets addressable by human recombinant antibodies |
| Grant number: |
Prot. BIRD187130 |
| Collection period: |
| From | To |
|---|
| 1 August 2018 | 31 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 |
| Last Modified: |
27 May 2022 08:19 |
|
orcid.org/0000-0003-3751-1691
