Cooperativity manifest in the binding properties of purified cardiac muscarinic receptors.
Muscarinic receptors were solubilized from porcine atria in digitonin-cholate and were purified by chromatography on DEAE-Sepharose and 3-(2'-aminobenzhydryloxy)tropane-Sepharose. The product identified on Western blots migrated with an apparent molecular mass of 60-75 kDa, with additional bands indicative of homotrimers (190 kDa) and homotetramers (240 kDa). Receptor eluted from the affinity column was accompanied by a mixture of guanyl nucleotide-binding proteins (G-proteins) identified on Western blots as Gi1/2, G(o), Gq/11, and Gs (preparation M2G); the G-proteins were largely removed by further processing on hydroxyapatite (preparation M2). Solubilized purified receptors bound muscarinic ligands in an apparently cooperative manner. In studies at equilibrium, the antagonists [3H]AF-DX 384, N-[3H]methylscopolamine (NMS), and [3H]quinuclidinylbenzilate (QNB) revealed Hill coefficients between about 0.8 and 1.2. Also, the apparent capacity for [3H]QNB exceeded that for [3H]AF-DX 384 and [3H]NMS by about 1.5-fold in M2 and by 2-fold in M2G. Binding to M2G at high concentrations of [3H]QNB was fully inhibited by unlabeled NMS, which therefore affected sites not labeled at similar concentrations of [3H]NMS. Oxotremorine-M displayed a biphasic inhibitory effect on the binding of [3H]AF-DX 384 in M2 and M2G, suggesting that multiple states of affinity are intrinsic to the receptor; 5'-guanylylimidodiphosphate was without appreciable effect in M2 but resulted in a bell-shaped binding profile for the agonist in M2G. All of the data can be described in terms of cooperative interactions within a receptor that is at least tetravalent and presumably an oligomer. In the context of the model, copurifying G-proteins and guanyl nucleotides serve to regulate the degree of cooperativity between successive equivalents of muscarinic ligands.
Wreggett
K A
KA
Faculty of Pharmacy, University of Toronto, Ontario, Canada.
Wells
J W
JW
D000494:Allosteric Regulation; D000818:Animals; D019204:GTP-Binding Proteins / Q000302:isolation & purification; D006165:Guanylyl Imidodiphosphate / Q000494:pharmacology; D066298:In Vitro Techniques; D008970:Molecular Weight; D018727:Muscarinic Antagonists; D009206:Myocardium / Q000378:metabolism*; D019832:N-Methylscopolamine; D010095:Oxotremorine / Q000378:metabolism; D010276:Parasympatholytics / Q000378:metabolism; D010890:Pirenzepine / Q000031:analogs & derivatives / Q000378:metabolism; D011813:Quinuclidinyl Benzilate / Q000378:metabolism / Q000494:pharmacology; D011869:Radioligand Assay; D011976:Receptors, Muscarinic / Q000302:isolation & purification / Q000378:metabolism*; D012508:Sarcolemma / Q000737:chemistry / Q000378:metabolism; D012602:Scopolamine Derivatives / Q000378:metabolism / Q000494:pharmacology; D013552:Swine
D018727:Muscarinic Antagonists; D010276:Parasympatholytics; D011976:Receptors, Muscarinic; D012602:Scopolamine Derivatives; C065959:AFDX 384; D006165:Guanylyl Imidodiphosphate; D010890:Pirenzepine; D010095:Oxotremorine; D011813:Quinuclidinyl Benzilate; D019204:GTP-Binding Proteins; D019832:N-Methylscopolamine