In mammals, sweet taste perception is mediated by the heterodimeric G-protein-coupled receptor, T1R2/T1R3. An interesting characteristic of this sweet taste receptor is that it has multiple ligand binding sites. Although there have been several studies on agonists of sweet taste receptors, little is known about antagonists of these receptors. In this study, we constructed a cell line stably expressing the human sweet taste receptor (hT1R2/hT1R3) and a functional chimeric G-protein (hG(alpha)16gust44) using the Flp-In system for measuring the antagonistic activity against the receptor. This constructed cell line responded quite intensely and frequently to the compounds applied for activation of hT1R2/hT1R3. In the presence of 3mM amiloride, the responses to sweet tastants such as sugar, artificial sweetener, and sweet protein were significantly reduced. The inhibitory activity of amiloride toward 1mM aspartame was observed in a dose-dependent manner with an IC(50) value of 0.87 mM. Our analysis of a cell line expressing hT1R3 mutants (hT1R3-A733V or hT1R3-F778A) made us to conclude that the target site of amiloride is distinct from that of lactisole, a known sweet taste inhibitor. Our results strongly indicate that amiloride reduces the sweet taste intensity by inhibiting the human sweet taste receptor and also that this receptor has multiple inhibitor binding sites.
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