Fig. 5.

Lys139Ile mutant binds inactive M₂R. COS-7 cells co-expressing M₂R-RLuc8 and indicated form of Venus-arrestin-3. Cells were pretreated for 5 min with vehicle or 10 μM inverse agonist atropine [50], and stimulated with carbamylcholine as in Fig. 2. Raw BRET ratios are shown. Basal arrestin-3 binding was not altered by atropine treatment. *, p < 0.05 (vs. vehicle control), analyzed with one-way repeated measures ANOVA, followed by Dunnett’s post-hoc test. Atropine prevented the effects of carbachol. #, p < 0.05 (significant interaction between the two treatments), analyzed with two-way repeated measures ANOVA, followed by Bonferroni post-hoc test.