Effects of N-ethylmaleimide on conformational equilibria in purified cardiac muscarinic receptors.
Muscarinic receptors purified from porcine atria and devoid of G protein underwent a 9-27-fold decrease in their apparent affinity for the antagonists quinuclidinyl benzilate, N-methylscopolamine, and scopolamine when treated with the thiol-selective reagent N-ethylmaleimide. Their apparent affinity for the agonists carbachol and oxotremorine-M was unchanged. Conversely, the rate of alkylation by N-ethylmaleimide, as monitored by the binding of [(3)H]quinuclidinyl benzilate, was decreased by antagonists while agonists were without effect. The receptor also underwent a time-dependent inactivation that was hastened by N-ethylmaleimide but slowed by quinuclidinyl benzilate and N-methylscopolamine. The destabilizing effect of N-ethylmaleimide was counteracted fully or nearly so at saturating concentrations of each antagonist and the agonist carbachol. Similar effects occurred with human M(2) receptors differentially tagged with the c-Myc and FLAG epitopes, coexpressed in Sf9 cells, and extracted in digitonin/cholate. The degree of coimmunoprecipitation was unchanged by N-ethylmaleimide, which therefore was without discernible effect on oligomeric size. The data are quantitatively consistent with a model in which the purified receptor from porcine atria interconverts spontaneously between two states (i.e. R R*). Antagonists favor the R state; agonists and N-ethylmaleimide favor the comparatively unstable R* state, which predominates after purification. Occupancy by a ligand stabilizes both states, and antagonists impede alkylation by favoring R over R*. Similarities with constitutively active receptors suggest that R and R* are akin to the inactive and active states, respectively. Purified M(2) receptors therefore appear to exist predominantly in their active state.
Department of Pharmacology and Faculty of Pharmacy, University of Toronto, Toronto, Ontario M5S 2S2, Canada.
Chi Shing
CS
Sum
Paul S-H
PS
Park
James W
JW
Wells
D000818:Animals; D015153:Blotting, Western; D002217:Carbachol / Q000494:pharmacology; D002460:Cell Line; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005033:Ethylmaleimide / Q000494:pharmacology*; D006801:Humans; D007313:Insecta; D007700:Kinetics; D008024:Ligands; D008956:Models, Chemical; D018721:Muscarinic Agonists / Q000494:pharmacology; D019832:N-Methylscopolamine / Q000494:pharmacology; D010095:Oxotremorine / Q000494:pharmacology; D010276:Parasympatholytics / Q000494:pharmacology; D011233:Precipitin Tests; D011485:Protein Binding; D011487:Protein Conformation / Q000187:drug effects; D011813:Quinuclidinyl Benzilate / Q000494:pharmacology; D043585:Receptor, Muscarinic M2; D011976:Receptors, Muscarinic / Q000737:chemistry* / Q000378:metabolism; D013439:Sulfhydryl Reagents / Q000494:pharmacology*; D013552:Swine; D013997:Time Factors
D004791:Enzyme Inhibitors; D008024:Ligands; D018721:Muscarinic Agonists; D010276:Parasympatholytics; D043585:Receptor, Muscarinic M2; D011976:Receptors, Muscarinic; D013439:Sulfhydryl Reagents; D010095:Oxotremorine; D011813:Quinuclidinyl Benzilate; D002217:Carbachol; D005033:Ethylmaleimide; D019832:N-Methylscopolamine