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 West ern 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 b y a mixture of guanyl nucleotide-binding proteins (G-proteins) identif ied on Western blots as G(i1/2), G(o), G(q/11), and G(s) (preparation M2G); the G-proteins were largely removed by further processing on hyd roxyapatite (preparation M2). Solubilized purified receptors bound mus carinic ligands in an apparently cooperative manner. In studies at equ ilibrium, the antagonists [H-3]AF-DX 384, N-[H-3]methylscopolamine (NM S), and [H-3]quinuclidinylbenzilate (QNB) revealed Hill coefficients b etween about 0.8 and 1.2. Also, the apparent capacity for [H-3]QNB exc eeded that for [H-3]AF-DX 384 and [H-3]NMS by about 1.5-fold in M2 and by S-fold in M2G. Binding to M2G at high concentrations of [H-3]QNB w as fully inhibited by unlabeled NMS, which therefore affected sites no t labeled at similar concentrations of [H-3]NMS. Oxotremorine-M displa yed a biphasic inhibitory effect on the binding of [H-3]AF-DX 384 in M 2 and M2G, suggesting that multiple states of affinity are intrinsic t o the receptor; 5'-guanylylimidodiphosphate was without appreciable ef fect in M2 but resulted in a bell-shaped binding profile for the agoni st in M2G. All of the data can be described in terms of cooperative in teractions within a receptor that is at least tetravalent and presumab ly an oligomer. In the context of the model, copurifying G-proteins an d guanyl nucleotides serve to regulate the degree of cooperativity bet ween successive equivalents of muscarinic ligands.