Figure 6: Recovery of Gi signalling of dualsteric probes in the allosteric M2 W4227.35A receptor mutant.
From: The allosteric vestibule of a seven transmembrane helical receptor controls G-protein coupling
(a,b) Equilibrium cell surface receptor binding of ACh, iperoxo, iper-6-phth and iper-6-naph competing against [3H]NMS in live CHO-hM2 cells (open symbols) and CHO-hM2 W4227.35A cells (filled symbols). Data are means±s.e.m. from at least three independent experiments conducted in triplicates. Binding curve fitting was based either on the four-parameter logistic function or on the allosteric ternary complex model in case of iper-6-phth wild type (WT) and iper-6-naph WT. Binding constants for the M2 WT receptor and M2 W422A mutant receptor were 5.86±0.04 and 4.36±0.02 for ACh, 7.49±0.16 and 6.81±0.01 for iperoxo, 6.11±0.06 and 5.45±0.09 for iper-6-phth and 7.07±0.08 and 6.05±0.04 for iper-6-naph, respectively. (c,d) M2 receptor-mediated Gi protein activation was measured as reflected by [35S]GTPγS binding to membranes of CHO-hM2 cells (open symbols) and CHO-hM2 W4227.35A cells (filled symbols). Maximum G-protein activation induced by ACh was set 100%. Data are means±s.e.m. from at least three independent experiments conducted in quadruplicates. Curve fitting in c and d was based on the operational model of agonism using the ligand-binding affinities derived from a and b. (e) Coupling efficiencies of indicated ligands at the M2 WT receptor and the M2 W4227.35A receptor mutant. (***P<0.001, *P<0.05), significantly different from iperoxo at the M2 WT receptor according to two-way ANOVA with Bonferroni's multiple comparison test, NS, not significantly different from iperoxo at the mutant receptors.
