Assay For Detecting G Alpha I-Mediated Decreases In Camp In Living Cells

Cell-based assays to detect G alpha i signaling are often indirect, frequently involve complex pharmacological interventions, and are usually blind to the kinetics of the signaling. Our goal was to develop a simple, direct measure of G alpha i signaling in living cells. We previously reported our fluorescent cADDis assay and showed that it reliably detects G alpha s-mediated increases in cAMP levels. Agonists that stimulate a Gs-coupled receptor produce changes in the intensity of bright green or red fluorescent protein sensors that can be followed over time using automated fluorescence plate readers or fluorescence imaging systems. Since the cADDis sensors can monitor G alpha s-mediated increases in adenylyl cyclase activity, in theory they should also be capable of detecting G alpha i-mediated decreases. Here we apply our green fluorescent cADDis sensor to the detection of G alpha i-mediated inhibition of adenylyl cyclase activity. We validated and optimized the assay in living HEK 293T cells using several known G alpha i-coupled receptors and agonists, and we report robust Z' statistics and consistent EC50 responses.