Our receptor assay described in this study was totally well correlated with this sensory data. In detail, NHDC or cyclamate at the concentration with same sweetness around 3% sucrose (i.e., 0.033 mM for NHDC and for 5.07 mM cyclamate) induces a synergistic enhancement of sweetness when each of them was added to 50 or 100 mM (i.e., 1.7% or 3.4%, respectively) sucrose solution. Accordingly, our data implies a strong association with the sweet intensity to the activation of sweet taste receptor, also indicating that the sweet enhancing of NHDC or cyclamate is just a result of the receptor-based synergisms. NHDC and cyclamate have been reported to interact with the

TMD of hT1R3 when they elicit the sweetness of their own, whereas aspartame, saccharin and acesulfame K are reported Protein Tyrosine Kinase inhibitor to bind to the VFTM of hT1R2 (Galindo-Cuspinera, Winnig, Bufe, Meyerhof, & Breslin, 2006). To examine whether the sweetness-potentiating effects of NHDC and cyclamate were derived from receptor–ligand interactions in the TMD of hT1R3, we utilised a cell line expressing mutant sweet-taste receptors, each with a point mutation in the hT1R3 TMD subunit (F778A and A733V). F778A was reported as a mutant with a reduced ability to recognise NHDC and cyclamate (Winnig et al., 2007). On the other Natural Product Library manufacturer hand, A733V does not affect the ability to recognise NHDC and cyclamate, and was then used as a positive

control here. We also examined the response of our stable cell lines to sucrose, NHDC and cyclamate, and confirmed the previously reported results with these mutations (Fig. S2A). Palmatine As shown in Fig. 3, the potentiating effect of NHDC or cyclamate was also observed when the cell line expressing the A733V mutant, as in the case with the WT receptor. In both cases, the cellular response to sucrose increased by the addition of 0.03 mM NHDC or 1 mM cyclamate. In contrast, the response of the F778A-expressing cells to sucrose was almost equal to the case that NHDC or cyclamate was added (Fig. 3). Furthermore, for the F778A mutant receptor, the enhancing effect of NHDC or

cyclamate was completely diminished to the case of 0.1 mM NHDC or 3 mM cyclamate (Fig. S2B). These observations suggest that the F778 residue in hT1R3 is critical for NHDC and cyclamate not only to activate the human sweet-taste receptor, but also to elicit synergistic potentiation to sucrose. Noting that the transmembrane domains six and seven are generally important for GPCR activation (Hu et al., 2005, Malherbe et al., 2003 and Petrel et al., 2003) and that their activation is often derived from conformational changes in the TMDs, our results proposed that this mechanism is probably true for hT1R activation. Interestingly, F778 in hT1R3 TM6 is also known as an essential site for the action of lactisole, which is a wide-acting inhibitor of the human sweet-taste receptor (Jiang, Cui, Zhao, Liu et al.