Using a radioalloster to test predictions of the cooperativity model for gallamine binding to the allosteric site of muscarinic acetylcholine M(2) receptors.

The muscarinic M(2) receptor contains an orthosteric and an allosteric site. Binding of an allosteric agent may induce a shift alpha of the equilibrium dissociation constant K(D) of 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 [(3)H]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 the K(A) shift induced for the prototype allosteric modulator gallamine by binding of N-methylscopolamine (NMS) to the orthosteric site of porcine heart M(2) receptors (4 mM Na(2)HPO(4), 1 mM KH(2)PO(4), pH 7.4; 23 degrees C; data are means +/- S.E.). First, in the common way, the concentration-dependent inhibition by gallamine of [(3)H]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 alpha = 46 (n = 5). The dissociation constant for gallamine binding at NMS-occupied receptors was predicted as p(alpha. K(A)) = 6.69. Labeling of the allosteric site by [(3)H]dimethyl-W84 allowed the measure of competitive displacement curves for gallamine. The K(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 microM NMS, to occupy the orthosteric site, gallamine displaced [(3)H]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 alpha = 46. These results validate the cooperativity model.