Figure 3. Evaluation of single or multiple point mutation affecting the human/squirrel monkey sweet taste receptors responses to sweeteners. A, HEK293E cells transiently transfected with human [hTas1R2/hTas1R3 (control)] and mutated hTas1R2 receptors along with Gα16-gust44. The responses of the cells to aspartame (10 mm), neotame (1 mm), and sucralose (2 mm) were assayed by calcium mobilization. The calcium response signals were normalized to the maximum response of hTas1R2/hTas1R3 receptor to sucralose (2 mm). Values represent the mean ± SEM of three independent experiments. The asterisk indicates significant differences tested by one-way ANOVA with Tukey's multiple-comparison test (*p < 0.05; **p < 0.01; ***p < 0.001) compared with the hTas1R2/hTas1R3 receptors. B, C, Dose–response of human and selected mutated human sweet taste receptors toward aspartame (B) and toward neotame (C). The responses of the cells to concentrations of the sweeteners aspartame and neotame were assayed by calcium mobilization. The calcium response signals were normalized to the maximum response of hTas1R2/hTas1R3 receptor to neotame. Mutants hTas1R2:S40T/D142E+hTas1R3, hTas1R2:I67S/D142E+hTas1R3, and hTas1R2:S40T/I67S/D142E+hTas1R3 showed no responses to aspartame versus good response of hTas1R2+hTas1R3 (EC50 = 5.0 × 10−3 m, B: 7.074, T: 73.32). Mutants hTas1R2:S40T/I67S/D142E+hTas1R3 (EC50 = 1.2 × 10−2 m, B: −1.486, T: 39.87), hTas1R2:S40T/D142E+hTas1R3 (EC50 = 1.4 × 10−3 m, B: 1.387, T: 61.12), and hTas1R2:I67S/D142E+hTas1R3 (EC50 = 4.7 × 10−3 m, B: −1.772, T: 51.81) showed weaker responses to neotame versus good response of hTas1R2+hTas1R3 (EC50 = 1.4 × 10−5 m, B: −165.1, T: 92.82)). The asterisks indicate significant differences tested by unpaired Student's t test (*p < 0.05; **p < 0.01; ***p < 0.001) compared with the hTas1R2/hTas1R3 receptors. D, HEK293E cells transiently transfected with squirrel monkey [smTas1R2/smTas1R3 (control)] and mutated smTas1R2 receptors along with Gα16-gust44. The responses of the cells to aspartame (10 mm), neotame (1 mm), and sucralose (2 mm) were assayed by calcium mobilization. The calcium response signals were normalized to the maximum response of hTas1R2/hTas1R3 receptor to sucralose (2 mm). Values represent the mean ± SEM of three independent experiments. The asterisk indicates significant differences tested by one-way ANOVA with Tukey's multiple-comparison test (**p < 0.01; ***p < 0.001) compared with the smTas1R2/smTas1R3 receptors. E, F, Dose–response of squirrel monkey, and selected mutated squirrel monkey sweet taste receptor toward aspartame (E) and toward neotame (F). The responses of the cells to concentrations of the sweeteners aspartame and neotame were assayed by calcium mobilization. The calcium response signals were normalized to the maximum response of hTas1R2/hTas1R3 receptor to neotame (B). Mutants smTas1R2:T40S/E142D+smTas1R3 (EC50 = 1.4 × 10−3 m, B: −3.314, T: 42.4), sm Tas1R2:S67I/E142D+smTas1R3 (EC50 = 5.6 × 10−3 m, B: −0.6682, T: 26.17), and smTas1R2:T40S/S67I/E142D+smTas1R3 (EC50 = 1.2 × 10−3 m, B: −2.882, T: 32.61) showed good response to aspartame versus no response of smTas1R2+smTas1R3. Mutants smTas1R2:T40S/S67I/E142D+smTas1R3 (EC50 = 2.8 × 10−4 m, B: −4.671, T: 51.73), smTas1R2:S67I/E142D+smTas1R3 (EC50 = 1.0 × 10−3 m, B: −5.754, T: 56.59), and smTas1R2:T40S/E142D+smTas1R3 (EC50 = 2.1 × 10−3 m, −4.24, T: 69.46) showed good responses to neotame versus no response of smTas1R2+smTas1R3. The asterisks indicate significant differences tested by unpaired Student's t test (***p < 0.001) compared with the smTas1R2/smTas1R3 receptors. Data (B, C, E, F) were fitted with sigmoid dose–response curves [Y = Bottom + (Top − Bottom)/(1 + 10LogEC50−X)] using GraphPad Prism software, and expressed as the mean ± SEM of the ΔF/F values from three independent experiments.