Cx43 GJ screening assay design

Assays to investigate cell-to-cell transfer of the second messenger cAMP through Cx43 and Cx32 GJs have been previously reported.^225-226 In these assays donor cells capable of generating cAMP via Gs protein-coupled receptor were co-cultured with biosensor cells harboring a cAMP-sensitive reporter – cAMP response element (CRE- luciferase reporter) – responding to cAMP by producing luminescence.^225-226 These assays despite their usefulness require disruption of the cells to be able to measure the cAMP-dependent activity of the CRE- luciferase reporter and do not monitor cAMP transfer in real-time. This method is tedious, costly, and less suitable for screening larger compound libraries for GJ modulators.

In the current project, a new strategy was developed to investigate the Cx43 GJs based on the transport of cAMP molecules from donor to biosensor cells. HeLa cells exogenously co-expressing the human adenosine receptor (A2AAR) and the mouse Cx43 were designed as donor cells, while cells co-expressing GloSensor luciferase (GSL) and mCx43 were created as biosensor cells.

The current assay design is based on the detection of (cAMP). It employs a genetically engineered firefly luciferase known as cAMP GloSensor-20F from Promega (GSL) which

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allows real-time detection of intracellular cAMP (Figure 33A). Another variant of this protein GloSensor-22F (GSL-22F) from Promega has been previously demonstrated to be useful for monitoring endogenous Gs protein-coupled receptors-induced cAMP generation in primary cells and HEK-293 cells co-cultured with biosensor cells harboring GSL-22F.²²⁷ This assay system is primarily developed to monitor GPCR activity in primary cells indirectly through biosensor cells connected via GJs, since transfection of primary cells is less efficient. This assay is based on endogenous connexins of primary neurons and HEK-293 cells, predominantly Cx32, and it does not report the background against other connexin subtypes.

Native firefly luciferase catalyzes the oxidation of its substrate luciferin in the presence of Mg²⁺, ATP and O2 to produce oxyluciferin, AMP, CO2 and luminescence signal (Figure 33B).

GSL is a circularly permuted form of firefly luciferase where between native N- and C-terminus, a conserved cAMP- binding domain B from PKA regulatory subunit type IIβ (RIIβB) is introduced. The binding of intracellular cAMP to GSL favors the functional conformation of luciferase which in turn metabolizes the luciferin containing cAMP GloSensor substrate (GSS) giving off yellow-green light.²²⁸

Figure 33: Principle of cAMP detection using engineered GloSensor luciferase (GSL). (A) GSL in the open conformation shows negligible luminescence activity, whereas binding of cAMP to (RIIβB), the cAMP binding site within GSL, favors the closed conformation and hence activates the luciferase, which metabolizes luciferin to produce luminescence. (B) Luciferase catalyzes the oxygenation of luciferin using molecular oxygen and ATP in the presence of Mg²⁺ to produce oxyluciferin, which is highly unstable in electronically excited state and produces light upon falling back to its ground state. Figure taken and modified from Fan et al. (2008).²²⁸

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According to the assay design the two cell populations will be co-cultured in a ratio of three donor cells to one biosensor cell for 4 h to allow the formation of GJs. The growth medium will then be replaced by the assay buffer supplemented with the luciferase substrate luciferin and the co-culture will be further incubated for 45 min in the dark at room temperature.

Thereafter, stimulation of the A2AAR by its selective agonist CGS21680 in the donor cells will activate adenylate cyclase, which converts ATP to cAMP. Binding of cAMP to GSL favors its functional conformation allowing it to metabolize luciferin and producing luminescence (see Figure 34). The mobilization of generated cAMP from donor to biosensor cells will take place via the heterologously expressed mCx43 GJs and could be detected in real-time by the GSL reaction in the established system. To initially evaluate the feasibility of the current assay design two preliminary experiments were performed:

i) Endogenous expression of adenosine receptors in HeLa cells: HeLa cells were selected for the development of a GJ assay due to low levels of endogenous Cx43 to maintain low background noise. In a preliminary study, the responses of endogenous Gs protein-coupled adenosine receptors, A2A and A2B, to non-selective and selective agonists in native HeLa cells were evaluated. HeLa cells were transiently transfected either with human A2AAR and GSL (HeLa-hA2A-GSL) or only with GSL (HeLa-GSL). HeLa-GSL produced a luminescence readout upon stimulation with forskolin (10 µM) that was used as an internal positive control.

However, activation of HeLa-GSL with A2AAR selective agonist CGS21680 (100 µM) produced a luminescence signal similar to vehicle (DMSO, 1%). A relatively moderate signal with HeLa-GSL was obtained with 50 µM NECA, a compound that is known to non-selectively activate A2A and A2B receptors. On the contrary, activation of HeLa-hA2A-GSL with either NECA (50 µM) or CGS21680 (100 µM) produced luminescence readouts even higher than those produced by forskolin (10 µM). These results indicated that generation of cAMP in HeLa-GSL cells in response to NECA can be primarily attributed to A2BARs, since activation by the A2AAR-selective agonist (CGS21680) did not generate detectable cAMP levels. In contrast, the high luminescence signal observed in HeLa-hA2A-GSL in response to CGS21680 was primarily due to activation of overexpressed A2AAR and that induced by NECA was due to activation of both, overexpressed A2AAR and endogenous A2BAR receptors.

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Based on this preliminary experiment, it was concluded that native HeLa cells do not or only weakly express A2AARs. It makes the A2AAR an excellent and selective cAMP generating tool using stimulation with the A2AAR-selective agonist (CGS21680) in HeLa cells against a nearly zero background. These results were in good agreement with the previously reported expression profile of A2A- and A2BARs in native HeLa cells, where A2BAR expression was found to be higher than that of A2AAR.²²⁹

ii) Effect of co-expression of proteins on luminescence signal: Co-expression of two or more proteins in the same cell may result in the reduced expression levels of each protein due to high occupancy of the cellular translational machinery. In the currently designed assay, biosensor cells transiently co-express GSL and mCx43. A comparison was therefore made between the transiently transfected HeLa cells expressing only GSL with the cells co-expressing GSL and mCx43 for their luminescence activity. Forskolin (10 µM) mediated activation of singly transfected HeLa cells with GSL gave an about 2-fold increased luminescence readout compared to the doubly transfected HeLa cells with GSL and mCx43. Similar results were obtained upon activation of both cell types with either 50 µM NECA or 50 µM adenosine with a 2-fold higher luminescence in singly transfected HeLa-GSL cells. Activation of both cell types by CGS21680 (10 & 100 µM) did not elicit luminescence signal indicating no expression of functional hA2AARs in native HeLa cells.

These preliminary results supported the concept that co-expression of Cx43 and GSL in HeLa cells appears to reduce the luminescence activity. Beside co-expression of proteins another reason could be that cAMP is released in the extracellular space through Cx43 hemichannels, since these channels are permeable to cAMP.²³⁰ However, luminescence readout in doubly transfected cells is still considerably above the basal luminescence, which indicates that the expressed amount of GSL is sufficient to detect changes in cellular cAMP levels.

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Figure 34: Design of the Cx43 GJ assay. HeLa cells expressing hA2AAR and mCx43 are denoted as donor cells and HeLa cells expressing GSL and mCx43 are indicated as biosensor cells. Both cell lines are co-cultured in a ratio 3:1 of donor to biosensor cells for 4 h to allow the formation of Cx43 GJs. The coupled cells are then equilibrated with buffer containing the substrate (luciferin) of engineered luciferase. Upon activation of Gs -coupled A2AARs, cAMP levels are elevated in the donor cells, cAMP molecules move to biosensor cells via Cx43 GJs formed between donor and biosensor cells. Once inside the biosensor cells, cAMP binds to GSL causing a conformational shift activating GSL, which creates a luminescence signal by oxidizing luciferin.