Fig. 4.
Representative phosphoinositide dose-response curves showing the action of ACh (•), NDMC (▿), AC-42 (▪), and 77-LH-28-1(⋄) on the wild-type M1 mAChR (a) and the following mutants: D105E (b), Y82A (c), L102A (d), Y408F (e), and W101A (f). The receptors were expressed in COS-7 cells labeled with [3H]inositol. Phosphoinositide response measurements were carried out using a scintillation proximity assay after 60 min of agonist stimulation. Points represent the mean ± S.E.M. of triplicate determinations. Curves show the fit to a four-parameter logistic function giving values for pEC50, EMax, EBasal, and a slope factor (usually approximately 0.9). EMax values were expressed as the percentage of the corresponding value for ACh (see Materials and Methods). Values are given as pEC50 (EMax) for ACh (•), NDMC (▿), AC-42 (▪), and 77-LH-28-1 (⋄), respectively. Values are designated N.D. if they could not be determined. Data shown are typical of three or more independent experiments. a, wild-type: 6.93 (100), 6.37 (42), 5.72 (67), 6.63 (73); b, D105E: 4.26 (100), N.D., N.D., N.D.; c, Y82A: 6.20 (100), 6.35 (36), N.D., N.D.; d, L102A: 4.75 (100), N.D., N.D., 5.40 (36); e, Y408F: 5.70(100), 6.59 (46), 4.64 (20), 5.33 (42); f, W101A: 5.49 (100), 6.12 (56), 7.46 (65), 8.70 (56).