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 ([Ca(2+)](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 S657(3.39)C, T780(6.44)A and M801(7.39)T disrupted the DFB-mediated increase in functional response. DFB-induced increase in potency was abolished in mutant receptors N733(45.51)A, Y791(6.55)A, A809(7.47)V, P654(3.36)S/S657(3.39)C and P654(3.36)S/S657(3.39)C/L743(5.47)V without affecting the enhancement of efficacy observed in wild type receptors. Mutations at positions Leu-743(5.47) and Trp-784(6.48) resulted in significantly larger DFB-induced potentiation of EC(50) and E(max) values than in wild type receptors. DFB-mediated increase of efficacy was abolished and EC(50) 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.