Figure 3. Cellular DMR reveals structure-dependent G_i bias of dualsteric probes.

(a–c) Shown are baseline-corrected DMR responses measured as wavelength shifts (pm) and recorded over time after addition of indicated concentrations of ACh in non-pretreated CHO-hM₂ cells (control, a) and either PTX (b)- or CTX (c)-pretreated CHO-hM₂ cells (means and s.e.m.) from one representative out of at least four independent experiments conducted in quadruplicates. (a) ACh exposure of control cells induces concentration-dependent positive DMR with peaks reflecting G_i signalling. (b) Pretreatment with the G_i inhibitor PTX (100 ng ml⁻¹, 16–24 h) reveals negative DMR reflecting G_s signalling. (c) Masking G_s-dependent signalling by CTX pretreatment (100 ng ml⁻¹, 8 h) induces positive DMR followed by sustained plateaus at higher concentrations. (d) Concentration-effect curves of ACh-induced peak DMR; data (means±s.e.m. from at least four experiments) are normalized to the response obtained with 100 μM ACh. The pronounced sensitivity of ACh's potency to PTX indicates G_i dominance in the control DMR peaks. (e–h) DMR recordings of indicated compounds in either control cells (e,g) or PTX-pretreated cells (f,h). Applied were maximally effective concentrations: ACh 100 μM, iper-6-phth 100 μM, iper-8-phth 10 μM, iperoxo 1 μM, iper-6-naph 100 μM, iper-8-naph 10 μM and iper-6 100 μM. Representative data (means and s.e.m.) of at least three independent experiments conducted in quadruplicates.