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.
Footnotes
- Received May 17, 1999.
- Accepted July 29, 1999.
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Send reprint requests to: Klaus Mohr, Department of Pharmacology and Toxicology, Institute of Pharmacy, University of Bonn, An der Immenburg 4, D-53121, Bonn, Germany. E-mail:K.Mohr{at}uni-bonn.de
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This work was supported by the Deutsche Forschungsgemeinschaft.
- The American Society for Pharmacology and Experimental Therapeutics
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Using a Radioalloster to Test Predictions of the Cooperativity Model for Gallamine Binding to the Allosteric Site of Muscarinic Acetylcholine M2 Receptors
