Structural Determinants of Allosteric Agonism and Modulation at the M4 Muscarinic Acetylcholine Receptor

Probe Dependence in the Allosteric Modulation of a G Protein-Coupled Receptor: Implications for Detection and Validation of Allosteric Ligand Effects

“…In contrast, when dissociation kinetic experiments were performed at the M 2 mAChR Y177A, no appreciable change in the dissociation rate of [ 3 H]NMS could be observed, supporting the hypothesis that mutation of the tyrosine in the E2L of the M 2 mAChR substantially reduces the binding of LY2033298. This latter finding is consistent with similar observations at the M 4 mAChR, whereby mutation of F186A (at the equivalent position in the E2L to Tyr177 at the M 2 mAChR) was identified as a key contributor to the binding affinity of LY2033298 for the allosteric site on that receptor (Nawaratne et al, 2010). Taken together, these radioligand binding experiments conclusively show that LY2033298 recognizes an allosteric site on the M 2 mAChR, in addition to its well characterized interaction with the M 4 mAChR.…”

“…Increasing concentrations of LY2033298 positively modulated the affinity of all the mAChR agonists studied but to markedly different extents, again indicating the occurrence of probe dependence. The effect of the modulator on ACh affinity was reasonable (␣ ϭ 16) but not as pronounced as we have previously noted at the M 4 mAChR (␣ values ranging up to 60) (Chan et al, 2008; Leach et al, 2010;Nawaratne et al, 2010;Leach et al, 2011;Suratman et al, 2011). Even lower degrees of positive cooperativity were observed with pilocarpine (2.8) and , whereas TMA (37) and xanomeline (36) were modulated to a greater extent than ACh.…”

“…Radioligand saturation binding and affinity parameters in these cells have been reported previously (Gregory et al, 2010). Interaction binding studies were also performed between [ 3 H]NMS, an IC 20 concentration of various mAChR agonists [ACh,oxotremorine,tetramethylammonium,pilocarpine,xanomeline,carbamoyloxy)-2-butynyltrimethylammnonium chloride (McN-A-343)], and increasing concentrations of LY2033298 by coincubating 20 g of M 2 mAChR FlpIn-CHO membrane in the presence of guanosine-5Ј-(␤␥-imino)triphosphate, as described previously (Nawaratne et al, 2010). For all experiments, nonspecific binding was defined by 10 M atropine, and the effects of vehicle were also determined.…”

“…In addition to demonstrating conclusively that LY2033298 interacts with the M 2 mAChR, our study has revealed that the binding of the modulator is very sensitive to mutation of Tyr177 within the E2L, a residue known to be important for the binding of prototypical modulators (Avlani et al, 2007;Valant et al, 2008). Given that mutation of Phe186 in the equivalent position of the M 4 mAChR E2L abolishes the binding of LY2033298 to that receptor (Nawaratne et al, 2010), as well as the fact that LY2033298 displays competitive behavior against the prototypical modulator C 7 /3-phth, at the M 4 mAChR , it is reasonable to speculate that the interaction of LY2033298 with the M 2 mAChR also involves, at least in part, binding to the site used by other well characterized mAChR modulators. We recently identified another conserved residue, Trp 3.28 , as contributing to the binding of LY033298 at the M 4 mAChR and proposed that this residue sits at the interface between orthosteric and allosteric sites ).…”

“…We have recently described the in vitro and in vivo pharmacological characteristics of a novel allosteric modulator, 3-amino-5-chloro-6-methoxy-4-methylthieno [2,3-b]pyridine-2-carboxylic acid cyclopropylamide (LY2033298), which is an allosteric agonist and a selective potentiator of the actions of ACh at the human M 4 mAChR but exhibits minimal interaction with the endogenous agonist at other mAChR subtypes (Chan et al, 2008;Nawaratne et al, 2008Nawaratne et al, , 2010Leach et al, 2010Leach et al, , 2011. It is noteworthy that when examining the potential for species variability in the actions of this modulator between human and rodent M 4 mAChRs, we found that the variability could be attributed to differences in the cooperativity between the modulator and ACh between species and is not due to differences in the allosteric binding pocket between the human and rodent receptors (Suratman et al, 2011).…”