Regulation of the CHAPS-solubilized muscarinic receptors by an inhibitory GTP binding protein (Gi) 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 Gi. This suggests that CHAPS solubilizes the mAChR-Gi complex both from neonatal and adult neurons. The gradient centrifugation of solubilized samples indicated that the neonatal samples contain mostly Gi-coupled mAChR (mAChRn) exhibiting a high affinity for [3H]OxM, but the adult samples contain a comparable quantity of Gi coupled mAChR (mAChRa1) and uncoupled mAChRs (mAChRa2) exhibiting low affinity for the agonist. The binding of [3H]OxM to or the dissociation of [3H]OxM from mAChRn and mAChRa1 was sensitive to GTP. However, the effects of GTP were sensitive to Na+ for mAChRn but not for mAChRa1. ADP-ribosylation by pertussis toxin but not by cholera toxin abolished the effects of Na+ on agonist binding to solubilized mAChRn, suggesting that NaCl affects mAChRn not by interacting with the receptor but by interacting with the mAChR-Gi complex. The subtype composition of mAChRn, mAChRa1 and mAChRa2 was studied by determining the IC50 and the Ki 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-mAChRn and M1-mAChRa1 was sensitive to GTP. The GTP-induced effects on mAChRn were modulated by Na+, but the effect on M1-mAChRa1 was not. This suggests functional differences between Gi coupled M1-mAChRn and M1-mAChRa1.
Department of Veterinary Diagnostic Medicine, College of Veterinary Medicine, University of Minnesota, St Paul 55108.
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Singh
D000375:Aging / Q000502:physiology*; D000818:Animals; D000831:Animals, Newborn; D001921:Brain / Q000378:metabolism*; D002793:Cholic Acids; D003902:Detergents; D019204:GTP-Binding Proteins / Q000502:physiology*; D066298:In Vitro Techniques; D008297:Male; D009474:Neurons / Q000378:metabolism; D011485:Protein Binding; D051381:Rats; D011976:Receptors, Muscarinic / Q000737:chemistry / Q000502:physiology*
D002793:Cholic Acids; D003902:Detergents; D011976:Receptors, Muscarinic; D019204:GTP-Binding Proteins; C028213:3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate