Regulation of the CHAPS-solubilized muscarinic receptors by an inhibitory GTP binding protein (G_i) in the brain of neonatal and adult rats

CHAPS-solubilized muscarinic receptors (mAChRs) retain selective agonist or antagonist binding and sensitivity to GTP and ADP-ribosylation by pertussis toxin that ribosylates a group of G protein called G_i. This suggests that CHAPS solubilizes the mAChR-G_i complex both from neonatal and adult neurons. The gradient centrifugation of solubilized samples indicated that the neonatal samples contain mostly G_i-coupled mAChR (mAChR_n,) exhibiting a high affinity for [³H]OxM, but the adult samples contain a comparable quantity of G_i coupled mAChR (mAChR_a1) and uncoupled mAChRs (mAChR_a2) exhibiting low affinity for the agonist. The binding of [³H]OxM to or the dissociation of [³H]OxM from mAChR_n and mAChR_a1 was sensitive to GTP. However, the effects of GTP were sensitive to Na⁺ for mAChR_n but not for mAChR_a1. ADP-ribosylation by pertussis toxin but not by cholera toxin abolished the effects of Na⁺ on agonist binding to solubilized mAChR_n, suggesting that NaCl affects mAChR_n not by interacting with the receptor but by interacting with the mAChR-G_i complex. The subtype composition of mAChR_n, mAChR_a1 and mAChR_a2 was studied by determining the ic₅₀ and the K_i values for the inhibition by subtype-selective antagonists in the binding of [³H]OxM to the receptors. The neonatal and adult samples exhibited differences in the distribution of the mAChR subtypes, the neonatal samples containing mostly the M₁-mAChR subspecies but the adult samples containing M₁-, M₂- and M₃-mAChR subspecies. The pirenzepine-induced inhibition in the binding of [³H]OxM both to M₁-mAChR_n and M₁-mAChR_a1 was sensitive to GTP. The GTP-induced effects on mAChR_n were modulated by Na⁺, but the effect on M₁-mAChR_a1 was not. This suggests functional differences between G_i coupled M₁-mAChR_n and M₁-mAChR_a1.