Depolarization-induced changes in the muscarinic receptor in rat brain and heart are mediated by pertussis-toxin-sensitive G-proteins

• Published in: The Journal of biological chemistry Vol. 266; no. 4; pp. 2595 - 2605
• Main Authors: M Cohen-Armon, M Sokolovsky
• Format: Journal Article
• Language: English
• Published: Bethesda, MD American Society for Biochemistry and Molecular Biology 05.02.1991
• Subjects: acetylcholine; Adenosine Diphosphate Ribose - metabolism; Adenosine Triphosphate - metabolism; Animals; Atropine - pharmacology; Biological and medical sciences; Brain - metabolism; Carbachol - pharmacology; Cell Membrane Permeability; Cell receptors; Cell structures and functions; Cells, Cultured; Chromatography, Thin Layer; Fundamental and applied biological sciences. Psychology; GTP-Binding Proteins - metabolism; guanine nucleotide-binding protein; Guanosine Diphosphate - metabolism; Guanosine Triphosphate - metabolism; heart; Male; Membrane Potentials; Molecular and cellular biology; Monoamines receptors (catecholamine, serotonine, histamine, acetylcholine); Myocardium - metabolism; Pertussis Toxin; Rats; Receptors, Muscarinic - metabolism; Receptors, Muscarinic - physiology; Synaptosomes - metabolism; Synaptosomes - physiology; Virulence Factors, Bordetella - pharmacology; Heart; Brain; Agonist; GTP; Radiolabelling; Rat; Rodentia; Permeabilizing; Activation; Chromatography; Depolarization; Binding protein; Vertebrata; Mammalia; Immunoblotting assay; Muscarinic receptor; G protein
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(18)52287-0

Muscarinic receptor properties in rat cortical and brain stem synaptoneurosomes and in heart myocytes were examined at resting potential and at depolarization. Depolarization induced the conversion of agonist-binding sites of the receptor from a high to a low affinity state, which could be reversed by a return to resting potential. No effect was observed on the affinity of the receptor for antagonists. Pertussis-toxin (PTX)-catalyzed ADP-ribosylation of all substrates in both synaptoneurosomal and myocyte membranes, when conducted at resting potential, prevented depolarization-induced conversion of the receptor affinity in these preparations. The target substrates were identified by [32P]ADP-ribosylation of membranes prepared from brain stem synaptoneurosomes. Autoradiography revealed labeling of a 39-kDa protein band, which reacted mainly with antibodies to the alpha-subunit of Go-proteins. The possible involvement of G-proteins in depolarization-induced changes in the receptor activity was further investigated by examining the effect of membrane potential on the PTX-sensitive binding of di- and triphosphated guanine nucleotides to synaptoneurosomal membranes. Brain stem synaptoneurosomes were made permeable to guanine nucleotides ([3H]GTP, [3H]GDP, [3H]5'-guanylyl imidodiphosphate) by treatment with ATP. After the synaptoneurosomes had been loaded with labeled GTP/GDP, resealed, and then subjected to either resting potential of short depolarization, binding of [3H]GDP to the membranes of depolarized synaptoneurosomes was 4.0 +/- 0.3 (n = 20) times higher than to the membranes of synaptoneurosomes at resting potential. Repolarization reversed this effect. Enhancement of [3H]GDP binding to the synaptoneurosomal membranes was induced also by muscarinic activation, although the increase obtained was only 30-40% (n = 5) relative to [3H]GDP binding at resting potential. Both the depolarization-induced and the muscarinically-induced enhancement of [3H]GDP binding were prevented following PTX-catalyzed ADP-ribosylation of G-proteins in the synaptoneurosomal membrane. Our results suggest that the depolarization-induced enhancement in the binding of [3H]GTP/[3H]GDP may be attributable to activation of PTX-sensitive G-proteins, which mediate the depolarization-induced alteration of the affinity of the muscarinic receptor for agonists.