Fig. 2
Development of R-GenGAR-DA1.2, which showed a negative red fluorescence response to DA. a Schematic illustration of a mutation site. Glutamine (Gln) was introduced into the N-terminal side of a linker in DA1.1. b Representative images of HeLa cells expressing DA1.2 treated with 10 µM DA, followed by 10 µM DRD1/5 antagonist SCH 23390 (SCH). Images are shown in the pseudocolor intensity-modulated display mode. c The fluorescence change (ΔF/F0) of DA1.2 in HeLa cells. Medium temperature was equilibrated before imaging (Additional file 1: Fig. S5b). DA (10 µM) and SCH (10 µM) were treated at time points indicated by pink and blue bars, respectively. Mean ΔF/F0 of 30 cells from 3 experiment are shown with SD (shaded area). Vehicle, 10 µM HCl or water; control, cells were only exposed to excitation light. d The dose–response curves, with temperature equilibration (Additional file 1: Fig. S4d), of DA (pink) and NE (green) in HeLa cells expressing DA1.2. DA: max ΔF/F0 = − 0.43 ± 0.01 and EC50 = 0.92 ± 0.13 µM; NE: max ΔF/F0 = − 0.43 ± 0.02 and EC50 = 61 ± 23 µM (DA, n = 8 experiments; NE, n = 10 experiments). Experimental data (dots) were fitted with the Hill equation (lines). DA1.2 has 66-fold selectivity for DA over NE. e Selectivity of DA1.2 for pharmacological compounds (n = 3–4 experiments, 10 cells per experiment; Additional file 1: Fig. S5c). All compounds = 10 µM. SKF 81297 (SKF), DRD1 agonist; haloperidol (Halo), DRD2 antagonist; Epi, epinephrine; 5-HT, serotonin; Glu, glutamate; GABA, γ-aminobutyric acid; His, histamine; Ach, acetylcholine; Oct, octopamine; Tyr, tyramine. For the vehicle condition, there was no significant difference between 10 µM HCl in H2O and 0.1% dimethyl sulphoxide (DMSO) (n = 4 experiments in each, 10 cells per experiment; Mann–Whitney U-test, p = 0.69). Therefore, these values were averaged and used as the vehicle condition. Mean ΔF/F0 values are shown with SEM. One-way ANOVA, F12,38 = 53.11, p < 0.0001; Dunnett's post hoc test (vs vehicle), ****p < 0.0001. f The on- and off-rate of DA1.2. The fluorescence intensity in each on- and off-kinetics was normalized by setting the maximum value as 0 and the minimum value as –1. The left panel indicates the time course of the normalized ΔF/F0 of DA1.2 (pink line) with the fitting curve (black dotted line). In the right panel, the on- and off-rates are shown as a box plot, in which the box shows the quartiles of data with the whiskers denoting the minimum and maximum, except for the outliers detected by 1.5 times the interquartile range (on-rate: 77 ± 5 ms, n = 13 cells; off-rate: 640 ± 160 ms, n = 9 cells)