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

Abstract

In the present study, we performed antibody-capture guanosine-5'-O-(3-[(35)S]thio)triphosphate ([(35)S]GTPγS) scintillation proximity assay (SPA), in which immuno-capture of Gα subunits following [(35)S]GTPγS binding was combined with SPA technology, in rat brain membranes. Preliminary experiments using a series of agonists and commercially available anti-Gα antibodies indicated the increase in specific [(35)S]GTPγS binding to Gα(q) determined with the anti-Gα antibody sc-393 and evoked by carbamylcholine chloride (CCh) was pharmacologically relevant. The experimental conditions were optimized as for the concentrations of GDP, MgCl(2), and NaCl, the dilution of the anti-Gα(q) antibody, and membrane protein contents incubated. Under the optimized conditions, CCh-stimulated specific [(35)S]GTPγS binding to Gα(q) in a concentration-dependent and saturable manner with an EC(50) of around 10 μM 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 [(35)S]GTPγS binding to Gα(q), even at a concentration as low as 1 nM. These results indicate that the functional coupling between M(1) mAChR and Gα(q) can be investigated in rat native brain membranes by means of antibody-capture SPA/[(35)S]GTPγS 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.