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Vol. 56, Issue 5, 962-965, November 1999

Using a Radioalloster to Test Predictions of the Cooperativity Model for Gallamine Binding to the Allosteric Site of Muscarinic Acetylcholine M2 Receptors

Christian Tränkle, Oliver Weyand, Alexandra Schröter, and Klaus Mohr

Department of Pharmacology and Toxicology, Institute of Pharmacy, University of Bonn, Bonn, Germany

The muscarinic M2 receptor contains an orthosteric and an allosteric site. Binding of an allosteric agent may induce a shift alpha  of the equilibrium dissociation constant KD of a radioligand for the orthosteric site. According to the cooperativity model, the KA 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 the KA shift induced for the prototype allosteric modulator gallamine by binding of N-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 pKA= 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  · KA) = 6.69. Labeling of the allosteric site by [3H]dimethyl-W84 allowed the measure of competitive displacement curves for gallamine. The Ki for gallamine at free receptors amounted to pKi,-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 pKi,+NMS = 6.60 ± 0.19 (n = 3). Thus, the NMS-induced pKi shift amounted to 47, which matches the predicted value of alpha  = 46. These results validate the cooperativity model.

Copyright © 1999 by The American Society for Pharmacology and Experimental Therapeutics


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