期刊
INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES
卷 22, 期 3, 页码 -出版社
MDPI
DOI: 10.3390/ijms22031417
关键词
compound library; connexin-43; gap junctions; GloSensor luciferase; HeLa cells; screening
资金
- German Federal Ministry of Education and Research (BMBF) [16GW0181K, 16GW0182]
Connexin gap junctions (Cx GJs) play a crucial role in cell-to-cell communication by allowing small molecules and ions to pass between cells. Dysfunction of Cx GJs is linked to diseases, and identifying modulators of Cx GJs may lead to new therapeutic drugs. This study established a novel assay using recombinant HeLa cells expressing Cx43 to screen for GJ modulators, showing potential for high-throughput screening.
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 A(2A) receptor, and biosensor cells expressing a cAMP-sensitive GloSensor luciferase were established. Adenosine A(2A) 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.
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