A Cellular Assay for the Identification and Characterization of Connexin Gap Junction Modulators
Open Access
- 31 January 2021
- journal article
- research article
- Published by MDPI AG in International Journal of Molecular Sciences
- Vol. 22 (3), 1417
- https://doi.org/10.3390/ijms22031417
Abstract
Connexin gap junctions (Cx GJs) enable the passage of small molecules and ions between cells and are therefore important for cell-to-cell communication. Their dysfunction is associated with diseases, and small molecules acting as modulators of GJs may therefore be useful as therapeutic drugs. To identify GJ modulators, suitable assays are needed that allow compound screening. In the present study, we established a novel assay utilizing HeLa cells recombinantly expressing Cx43. Donor cells additionally expressing the Gs protein-coupled adenosine A2A receptor, and biosensor cells expressing a cAMP-sensitive GloSensor luciferase were established. Adenosine A2A receptor activation in the donor cells using a selective agonist results in intracellular cAMP production. The negatively charged cAMP migrates via the Cx43 gap junctions to the biosensor cells and can there be measured by the cAMP-dependent luminescence signal. Cx43 GJ modulators can be expected to impact the transfer of cAMP from the donor to the biosensor cells, since cAMP transit is only possible via GJs. The new assay was validated by testing the standard GJ inhibitor carbenoxolon, which showed a concentration-dependent inhibition of the signal and an IC50 value that was consistent with previously reported values. The assay was demonstrated to be suitable for high-throughput screening.Funding Information
- Bundesministerium für Bildung und Forschung (CONNEXIN - 16GW0181K and 16GW0182)
This publication has 42 references indexed in Scilit:
- A High-Throughput Assay for Connexin 43 (Cx43,GJA1) Gap Junctions Using Codon-Optimized AequorinASSAY and Drug Development Technologies, 2013
- Cyclic nucleotide permeability through unopposed connexin hemichannelsFrontiers in Pharmacology, 2013
- A novel mutation in the connexin 26 gene (GJB2) in a child with clinical and histological features of keratitis–ichthyosis–deafness (KID) syndromeClinical and Experimental Dermatology, 2011
- The Danger Signal Adenosine Induces Persistence of Chlamydial Infection through Stimulation of A2b ReceptorsPLOS ONE, 2009
- Connexins and their channels in cell growth and cell deathCellular Signalling, 2006
- Life Cycle of Connexins: Regulation of Connexin Synthesis and DegradationAdvances in cardiology, 2006
- Defective Epidermal Barrier in Neonatal Mice Lacking the C-Terminal Region of Connexin43Molecular Biology of the Cell, 2004
- A Simple Statistical Parameter for Use in Evaluation and Validation of High Throughput Screening AssaysSLAS Discovery, 1999
- Cyclic Stretch Enhances Gap Junctional Communication Between Osteoblastic CellsJournal of Bone and Mineral Research, 1998
- Scrape-loading and dye transfer: A rapid and simple technique to study gap junctional intercellular communicationExperimental Cell Research, 1987