Cholinergic muscarinic receptor in synaptosomal membranes. Heterogeneity of binding sites for l-[³H]quinuclidinyl benzilate

We studied the binding of radioactive L-quinuclidinyl benzilate (L-[³H]QNB) to muscarinic cholinergic receptor sites of synaptosomal membranes of cat cerebral cortex. The experimental data were analyzed with the use of the Fortran IV program NLIN2, in an IBM/360 computer system DOS. The data obtained were compared with the model for n number of heterogeneous sites (n=1, 2, or 3), and also with a model for cooperative bivalent sites. The best fitting was obtained with the two-population site model (K(D1) = 5.2 and K(D2) = 144 pm; B(max1) = 366 and B(max2) = 558 pmoles/g of protein) when the data were analyzed by the Scatchard equation (p<0.01 according to Fischer's F-test). Competition with the muscarinic antagonist atropine and scopolamine gave IC₅₀ values, respectively, of 1.4 and 3.4, and Hill coefficient of about 1. Competition with the muscarinic agonist oxotremorine and with carbamylcholine gave IC₅₀ values, respectively, of 0.9 and 47 μM, and the Hill coefficients were 0.70 and 0.56. Consistent with the heterogeneity of sites found by equilibrium binding, heterogeneity was also observed in experiments of kinetic binding. The dissociation was biphasic, with a rapid component having k(-2) = 37.7 x 10^-3 min^-1, and a slow component having k(-1) = 4.8 x 10^-3 min^-1. The two-site model gave a much better fitting of the dissociation data than did the one-site model (p<0.01, F-test). Because of the equilibrium and dissociation results, the association data were analyzed by the two-site model. A fast association component (k(+1) = 4.9 x 10^-4 pmoles^-1 min^-1) and a slow association component (k(+2) = 1.1 x 10^-4 pmoles^-1 min^-1) were found. The constants calculated from kinetic data for the two-site model were K(D1) = 9.7 pm and K(D2) = 342 pm, which showed little difference with those obtained by equilibrium binding. The results suggest the existence of two independent site populations for L-[³H]QNB binding in synaptosomal membranes of the cat cerebral cortex.