Figure 7

Inhibition of agonist-induced Ca²⁺ signaling by expressed mRFP-Tubby-domain, PLCδ1PH-mRFP and p130-PH-mRFP. COS-7 cells were transfected with the indicated mRFP fusion constructs, and their cytosolic Ca²⁺ responses were analyzed by ratiometric Ca²⁺ imaging using Fura2. The amount of fluorescent protein was determined after the release of all membrane bound constructs by the addition of high concentraction of ionomycin at the end of each experiment. ATP-induced Ca²⁺ responses were grouped into four categories according to expression levels of the red fluorescence and the responses were averaged (see Table 2 for these values in each group in arbitrary units). Note that each construct interferes with the Ca²⁺ signal in these COS-7 cells where the ATP-induced PLC activation and InsP₃ increase is relatively modest. Such inhibitory effects are substantially smaller in HEK293-AT1 cells that show robust PLC activation (see Fig. 8). Table 2 shows the numerical values of the Ca²⁺ signaling parameters.