Determination of key amino acids implicated in the actions of allosteric modulation by 3,3′-difluorobenzaldazine on rat mGlu5 receptors

Several mutations in the seven-transmembrane region of rat metabotropic glutamate 5 (rmGlu5) receptors were produced by site-directed mutagenesis and expressed in CHO cells. Using functional intracellular calcium ([Ca2+]i) mobilisation, we identified amino acids implicated in the positive allosteric modulation of quisqualate-induced response by 3,3′-difluorobenzaldazine (DFB). Human and rat mGlu5 receptors displayed a higher potency and a higher efficacy in the presence of DFB. Mutant receptors S6573.39C, T7806.44A and M8017.39T disrupted the DFB-mediated increase in functional response. DFB-induced increase in potency was abolished in mutant receptors N73345.51A, Y7916.55A, A8097.47V, P6543.36S/S6573.39C and P6543.36S/S6573.39C/L7435.47V without affecting the enhancement of efficacy observed in wild type receptors. Mutations at positions Leu-7435.47 and Trp-7846.48 resulted in significantly larger DFB-induced potentiation of EC50 and Emax values than in wild type receptors. DFB-mediated increase of efficacy was abolished and EC50 values were right-shifted in mutant receptor F787A, resulting in DFB acting as a weak partial antagonist at this mutant receptor. Based on these findings, we constructed a homology model concluding that six key residues in transmembranes 3, 5, 6 and 7 are necessary for the allosteric modulation of rmGlu5a receptor by DFB. The model confirms an overlapping but distinct binding site to 2-methyl-6-(phenylethynyl)-pyridine (MPEP), and in particular emphasises the key role of W784 in transmembrane (TM) 6 for controlling the receptor's activation state.