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Fig. 6. All functional combinations of TIRs and chimeras support our model (Fig. 5). (A) All 16 combinations of T1Rs and chimeras were tested for responses to sweeteners and lactisole. One of the combinations, rat T1R2/T1R3H-R, appears nonfunctional as it generated no response to any sweeteners tested. Two other combinations, rat T1R2/human T1R3, and T1R2H-R/T1R3R-H, generated very low response to sucrose and AceK, which make it impossible for us to determine the inhibition effect of lactisole on those responses. However, their responses to cyclamate were significant and can be inhibited by lactisole (data not shown). T1R chimeras were transiently transfected into HEK-293T cells with Ga _15/i1. The activities represent the mean ± SE of number of responding cells for four imaged fields of ~1,000 confluent cells; each unit on the y axis represents 50 responding cells. Suc, sucrose 100 mM; Suc/Lac, sucrose 100 mM, lactisole 1 mM; AceK, 10 mM; AceK/Lac, AceK 10 mM, lactisole 1 mM; ATM, aspartame 10 mM; NTM, neotame 10 mM; Cyc, cyclamate 10 mM. (B) The lactisole dose-dependent inhibition curves of the human sweet receptor were determined for sucrose (Suc), saccharin (Sac), and D-tryptophan (D-Trp), each at two different concentrations. The IC₅₀s are 19.6 ± 0.1 and 64.6 ± 0.3 m M for sucrose at 50 and 120 mM, respectively; 22.6 ± 0.1 and 103 ± 7 m M for saccharin at 0.1 and 2 mM, respectively; and 19.9 ± 0.2 and 168 ± 9 m M for D-tryptophan. (C) The dose-responses of human sweet receptor for sucrose, D-Trp, and saccharin were determined with different concentrations of lactisole. In the presence of 0, 10, or 20 m M lactisole, the EC₅₀s were 19.4 ± 0.9, 24.7 ± 1.0, and 31.3 ± 0.3 mM, respectively, for sucrose; 0.37 ± 0.02, 0.60 ± 0.03, and 0.94 ± 0.08 mM, respectively, for D-Trp; and 42 ± 3, 67 ± 6, and 118 ± 2 m M, respectively, for saccharin. Values represent the mean ± SE for four independent responses. The dose-responses in B and C were determined at least six times independently and generated similar results as shown here.