Abstract

The muscarinic M₂ receptor contains an orthosteric and an allosteric site. Binding of an allosteric agent may induce a shift α of the equilibrium dissociation constant K _Dof a radioligand for the orthosteric site. According to the cooperativity model, the K _A of alloster binding is expected to be shifted to an identical extent depending on whether the orthosteric site is occupied by the orthoster or not. Here, the novel radioalloster [³H]dimethyl-W84 (N,N′-bis[3-(1,3-dihydro-1,3-dioxo-4-methyl-2H-isoindol-2-yl)propyl]-N,N,N′,N′-tetramethyl-1,6-hexanediaminium diiodide) was applied to directly measure theK _A shift induced for the prototype allosteric modulator gallamine by binding ofN-methylscopolamine (NMS) to the orthosteric site of porcine heart M₂ receptors (4 mM Na₂HPO₄, 1 mM KH₂PO₄, pH 7.4; 23°C; data are means ± S.E.). First, in the common way, the concentration-dependent inhibition by gallamine of [³H]NMS equilibrium binding was measured and analyzed using the cooperativity model, which yielded for the affinity of gallamine binding at free receptors a pK _A= 8.35 ± 0.09 and a cooperativity factor α = 46 (n = 5). The dissociation constant for gallamine binding at NMS-occupied receptors was predicted as p(α · K _A) = 6.69. Labeling of the allosteric site by [³H]dimethyl-W84 allowed the measure of competitive displacement curves for gallamine. TheK _i for gallamine at free receptors amounted to pK _i,−NMS = 8.27 ± 0.39 (n = 5), which is in line with the prediction of the cooperativtiy model. In the presence of 1 μM NMS, to occupy the orthosteric site, gallamine displaced [³H]dimethyl-W84 with pK _i,+NMS = 6.60 ± 0.19 (n = 3). Thus, the NMS-induced pK _i shift amounted to 47, which matches the predicted value of α = 46. These results validate the cooperativity model.