In humans, the 25 bitter taste receptors (T2Rs) are activated by hundreds of structurally diverse bitter compounds. However, only 5 antagonists or bitter blockers are known. In this study, using molecular modeling guided site-directed mutagenesis we elucidated the ligand binding pocket of T2R4. We found seven amino acids located in the extracellular side of transmembrane 3 (TM3), TM4, extracellular loop 2 (ECL2), and ECL3 to be involved in T2R4 binding to its agonist quinine. ECL2 residues Asn173 and Thr174 are essential for quinine binding. Guided by a molecular model of T2R4 a number of amino acid derivatives were screened for their ability to bind to T2R4. These predictions were tested by calcium imaging ass... More
In humans, the 25 bitter taste receptors (T2Rs) are activated by hundreds of structurally diverse bitter compounds. However, only 5 antagonists or bitter blockers are known. In this study, using molecular modeling guided site-directed mutagenesis we elucidated the ligand binding pocket of T2R4. We found seven amino acids located in the extracellular side of transmembrane 3 (TM3), TM4, extracellular loop 2 (ECL2), and ECL3 to be involved in T2R4 binding to its agonist quinine. ECL2 residues Asn173 and Thr174 are essential for quinine binding. Guided by a molecular model of T2R4 a number of amino acid derivatives were screened for their ability to bind to T2R4. These predictions were tested by calcium imaging assays which led to identification of γ-aminobutryic acid (GABA) and Nα,Nα-Bis(carboxymethyl)-L-lysine (BCML) as competitive inhibitors of quinine activated T2R4 with an IC50 of 3.2 ± 0.3 μM and 59 ± 18 nM, respectively. Interestingly, pharmacological characterization using a constitutively active mutant of T2R4 reveals that GABA acts as an antagonist, while BCML acts as an inverse agonist on T2R4. Site-directed mutagenesis confirms that the two novel bitter blockers share the same orthosteric site as the agonist quinine. The signature residues Ala90 and Lys270 play important roles in interacting with BCML and GABA, respectively. This is the first report to characterize a T2R endogenous antagonist and an inverse agonist. The novel bitter blockers will facilitate physiological studies focused on understainding the roles of T2Rs in extra-oral tissues.
