Depolarization-induced changes in the muscarinic receptor in rat brain and heart are mediated by pertussis-toxin-sensitive G-proteins |
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Authors: | M Cohen-Armon M Sokolovsky |
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Institution: | Department of Biochemistry, George S. Wise Faculty of Life Sciences, Tel Aviv University, Israel. |
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Abstract: | 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. |
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