FIGURE 6.

Mutation of Y177A in the allosteric pocket affects the efficacy of McN-A-343 and the potency of DDBL-4 and changes the allosteric binding mode of DDBL-4 at the human M₂ mAChR. A, concentration-response curves to ACh, TMA, and McN-A-343 for the stimulation of ERK1/2 phosphorylation. Data points were determined by taking the time to peak response (5 min) for each agonist concentration at 37 °C in CHO-FlpIn cells stably expressing human M₂ Y177A mutant mAChR. Data represent the mean ± S.E. obtained from four experiments conducted in duplicate. Also shown in the dashed curve is the response of McN-A-343 at the wild type (WT) receptor (taken from Fig. 4). B, concentration-effect relationships for DDBL-4 on the dissociation rate of [³H]NMS (0.5 nm; 37 °C) at the wild type or Y177A M₂ mAChR stably expressed in membranes from CHO-FlpIn cells. Data represent the mean ± S.E. obtained from three experiments conducted in triplicate; data points for the wild type receptor are taken from Fig. 3 (lower). C, ACh- or TMA-mediated ERK1/2 phosphorylation in the absence (solid symbols) or presence (open symbols) of 100 μm DDBL-4 for 5 min at 37 °C in CHO-FlpIn cells stably expressing the Y177A mutant M₂ mAChR. Data represent the mean ± S.E. obtained from six experiments conducted in duplicate.