Responses of cells expressing various γ subunits to different concentrations of agonist.
A. M3-CHO cells expressing αq-CFP, YFP-γ (as indicated) and PH-mCh were imaged as mentioned in Methods and Text S1. Representative images of cells exposed to 1 µM carbachol (agonist) are shown. The decrease in YFP-γ3 intensity observed is due to bleaching during acquisition. B. Translocation of PH-mCh at specific agonist concentrations. Bars show changes for PH-mCh translocation in the presence of various γ subunits. No. of γ3 cells examined at 250 nM–20; at 1 µM–11; at 5 µM–17 and at 100 µM–17. No. of γ9 cells examined at 250 nM–23; at 1 µM–11; at 5 µM–17 and at 100 µM–14. Cells with comparable expression of PH-mCh were imaged. Regions in the cytosol were selected for analysis (as indicated by circles). Values were calculated as ratio of M-B/B, where M is the maximal intensity at a particular concentration and B, the intensity before exposure to that concentration of agonist. Results are the means ± SEM. *p<0.01, **p<0.001. C. Translocation of YFP-γ9 subunits at specific agonist concentrations. Bar diagrams show changes in YFP-γ9 emission on the plasma membrane (N = 4). Regions on the plasma membrane were selected for analysis (as indicated by arrows in image, Fig. 1A, right panel). Values are shown as percentile of means ± SEM. *p<0.01, (response to 250 nM and 100 µM to others).