Muscarinic acetylcholine receptor-mediated activation of Gq in rat brain membranes determined by guanosine-5'-O-(3-[35S]thio)triphosphate ([35S]GTPγS) binding using an anti-G protein scintillation proximity assay

In the present study, we performed antibody-capture guanosine-5'-O-(3-[S-35]thio)triphosphate ([S-35]GTP gamma S) scintillation proximity assay (SPA), in which immuno-capture of G alpha subunits following [S-35]GTP gamma S binding was combined with SPA technology, in rat brain membranes. Preliminary experiments using a series of agonists and commercially available anti-G alpha antibodies indicated the increase in specific [S-35]GTP gamma S binding to G alpha(q) determined with the anti-G alpha antibody sc-393 and evoked by carbamylcholine chloride (CCh) was pharmacologically relevant. The experimental conditions were optimized as for the concentrations of GDP, MgCl2, and NaCl, the dilution of the anti-G alpha(q) antibody, and membrane protein contents incubated. Under the optimized conditions, CCh-stimulated specific [S-35]GTP gamma S binding to G alpha(q) in a concentration-dependent and saturable manner with an EC50 of around 10 mu M in all of the membranes prepared from rat hippocampus, cerebral cortex, and striatum. The maximum responses were varied according to the brain regions, with the rank order in magnitude of hippocampus > cerebral cortex > striatum. The addition of MT-7, a snake toxin with high selectivity for M-1 over the other muscarinic acetylcholine receptors (mAChRs) (M-2-M-5), almost completely extinguished CCh-stimulated [S-35]GTP gamma S binding to G alpha(q), even at a concentration as low as 1 nM. These results indicate that the functional coupling between M-1 mAChR and G alpha(q) can be investigated in rat native brain membranes by means of antibody-capture SPA/[S-35]GTP gamma S binding assay. The assay developed in the present study would provide a useful strategy for investigation of possible pathophysiological alterations in neuropsychiatric disorders such as Alzheimer's disease and schizophrenia as well as for drug discovery.