Supporting Information Figure 1. Kinetics of aequorin and aequorinN26D responses compared to those of GA and GAN26D in vitro. A1-A3. Time course of light responses from aequorin (grey line) and GA (dark line) after mixing with Ca2+ 1 mM (A1), Ca2+ 1 mM + Mg2+ 1 mM (A2) and Ca2+ 1 mM + Mg2+ 2 mM (A3). Inset shows magnified view of the rise phase kinetics. Each trace is the average of at least 20 single trials and is normalized to the peak intensity. Sampling rates were 2 kHz for the first second followed by 200 Hz. Note that GA responses decayed faster than aequorin responses. B1-B3. Comparison of aequorinN26D and GAN26D. Same protocol as in A.
Supporting Information Figure 2. Estimation of intracellular concentrations of GA, GO and GAN26D expressed in layer V pyramidal neurons with two-photon microscopy. A. A fluorescence calibration curve was obtained with fluorescein solutions of known concentrations at pH 9. Squares represent experimental values measured from four different ROI, which were fitted with linear functions (solid lines). Note that experimental values were almost superimposed, as well as linear fits. B. Left. Single plane image of a GO-expressing layer V pyramidal neuron, where fluorescence intensity was measured at the cytoplasmic ROI delineated. Right. GFP-photoprotein cytosolic concentrations measured in GA- (n=21 cells), GO- (n=26 cells), and GAN26D-expressing (n=13 cells) neurons. Data are expressed as mean ĂÄ standard deviation.