The intrinsic activity of agonists to activate M₁ and M₃ mACh receptors stably expressed in CHO cells was assessed using two [³⁵S]-GTPγS binding methodologies. The total [³⁵S]-GTPγS binding assay measures the global activation of the cellular G protein population, however, through the use of pertussis toxin (PTx), to prevent receptor-mediated activation of G_i-like G subunits, inferences about the subclasses of G proteins activated can be made. Agonist-G protein activation profiles were further investigated using an antibody-capture technique (Akam et al., 2001), which permits the potency or intrinsic activity of G_q/11 and G_il-3 subunit activation in to be assessed.

Concentration-response curves constructed for total [³⁵S]-GTPγS binding stimulated by methacholine (MCh) in CHO-m1 membranes were biphasic comprising high- and low-affinity components (pEC₅₀s high, 6.23 ± 0.28; low, 4.33 ± 0.05). Pilocarpine failed to cause a significant increase in total [³⁵S]-GTPγS binding. Following pre-treatment of CHO cells with PTx (100 ng ml^-1, 24 h), monophasic increases in [³⁵S]-GTPγS binding stimulated by agonist were observed; with the EC₅₀ correlating with the high affinity binding seen in control CHO-m1 membranes (pEC₅₀, 6.26 ± 0.06). Immunoprecipitation of G_q/11 following [³⁵S]-GTPγS binding yielded a similar pEC₅₀ estimate (6.21 ± 0.13) for the MCh-stimulated response, supporting the hypothesis that the M₁ mACh receptor is efficiently couples to G_q/11 . The improved sensitivity of the antibody-capture method revealed that pilocarpine robustly stimulated G_q/11 -[³⁵S]-GTPγS binding (pEC₅₀, 6.00 ± 0.17). Assessment of G_il-3 -[³⁵S]-GTPγS binding revealed that only the most efficacious agonists could elicit responses above basal, with pilocarpine ineffective. Thus, agonist activation of the M₁ mACh receptor appears to follow a ‘strength-of-signal’ model, where agonists activate G_q/11 proteins most effectively, only high efficacy agonists active the relatively poorly-coupled G_il-3 proteins.

Total [³⁵S]-GTPγS binding concentration-response curves constructed for agonist-mediated responses in CHO-m3 membranes were shallow, but could not reproducibly be better fitted to a two-site model. Following PTx treatment, maximal agonist-mediated total [³⁵S]-GTPγS binding responses were reduced indicating again that a heterogeneous population of G proteins is activated, but that the potency differences for PTx-sensitive and -insensitive G proteins is too small to allow their EC₅₀s to be determined. In agreement, the antibody capture method revealed that a subset of mACh receptor agonists could stimulate robust [³⁵S]-GTPγS binding to both G_q/11 and G_il-3 proteins. Although pilocarpine did not stimulate a significant increase in [³⁵S]-GTPγS-G_q/11 binding it did increase [³⁵S]-GTPγS-G_il-3 binding to approx. 50% of the response evoked by MCh. Therefore, with respect to the M₃ mACh receptor, our data provides some evidence for agonist-specific conformations with different G subunit activation profiles.

Akam, E.C. et al. (2001) Br. J. Pharmacol., 132, 950-958.