Identification of drugs competing with d-tubocurarine for an allosteric site on cardiac muscarinic receptors.
d-Tubocurarine behaved as a weak allosteric inhibitor of N-[3H] methylscopolamine binding to cardiac M2 muscarinic receptors. In a low ionic strength buffer devoid of bivalent ions, d-tubocurarine recognized cardiac M2 receptors in the micromolar concentration range and decreased their affinity for N-[3H]methylscopolamine by at most 4-fold. To identify the compounds that preferentially recognize this accessory site (as opposed to the classical muscarinic binding site), we measured the inhibition by different drugs of N-[3H]methylscopolamine binding, in the absence or presence of d-tubocurarine. The effect of gallamine was competitively inhibited by d-tubocurarine; both drugs compete for the same accessory site on muscarinic receptors. The effects of dexetimide, levetimide, 4-diphenylacetoxy-N-ethylpiperidine ethobromide, AF-DX 116, and telenzepine on N-[3H]methylscopolamine binding were not affected or were barely affected by d-tubocurarine; these compounds preferentially recognize another binding site (probably the muscarinic binding site). The dose-effect curves for pentamethylene-bis(4-diphenylacetoxymethylpiperidine) bromide and methoctramine were shifted, but at most 10-fold, by d-tubocurarine. It is likely that (in this low ionic strength incubation buffer) methoctramine and pentamethylene-bis(4-diphenylacetoxymethylpiperidine)bromide had comparable affinities for the muscarinic site and the accessory site. d-Tubocurarine competitively inhibited their binding to the accessory site and allosterically inhibited their binding to the muscarinic site. This resulted in a large decrease (40-60-fold) of their overall affinity for muscarinic receptors.
Department of Biochemistry and Nutrition, Medical School, Université Libre de Bruxelles, Belgium.
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Waelbroeck
D000495:Allosteric Site; D000818:Animals; D001667:Binding, Competitive; D008297:Male; D009206:Myocardium / Q000378:metabolism*; D019832:N-Methylscopolamine; D051381:Rats; D017208:Rats, Wistar; D011976:Receptors, Muscarinic / Q000378:metabolism*; D012602:Scopolamine Derivatives / Q000037:antagonists & inhibitors; D014403:Tubocurarine / Q000378:metabolism*
D011976:Receptors, Muscarinic; D012602:Scopolamine Derivatives; D019832:N-Methylscopolamine; D014403:Tubocurarine