Figure 2.

Design of the Cx43 GJ assay. HeLa cells expressing A_2AAR and Cx43 are denoted as donor cells and HeLa cells expressing GloSensor luciferase and Cx43 as biosensor cells. The cell lines were cocultured in a ratio of 3:1 (donor: biosensor cells) for 4 h to allow the formation of Cx43 GJs. The cells were then equilibrated with buffer containing the substrate (luciferin) of the engineered cAMP-dependent luciferase. Upon activation of the G_s protein-coupled A_2AARs, cAMP levels in the donor cells are increased. cAMP can then migrate via the Cx43 GJs to the biosensor cells. There, cAMP binds to the GloSensor luciferase which results in a conformational change that leads to an activation of the GloSensor luciferase, creating a luminescence signal by oxidation of luciferin. Depiction of GloSensor luciferase is based on reference [25].