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

• Published in: Molecular pharmacology Vol. 56; no. 5; p. 962
• Main Authors: Christian Tränkle, Oliver Weyand, Alexandra Schröter, Klaus Mohr
• Format: Journal Article
• Language: English
• Published: American Society for Pharmacology and Experimental Therapeutics 01.11.1999
• ISSN: 0026-895X; 1521-0111
• DOI: 10.1124/mol.56.5.962

The muscarinic M 2 receptor contains an orthosteric and an allosteric site. Binding of an allosteric agent may induce a shift α 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-2 H -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°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 p K 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 [ 3 H]dimethyl-W84 allowed the measure of competitive displacement curves for gallamine. The K i for gallamine at free receptors amounted to p K 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 [ 3 H]dimethyl-W84 with p K i,+NMS = 6.60 ± 0.19 ( n = 3). Thus, the NMS-induced p K i shift amounted to 47, which matches the predicted value of α = 46. These results validate the cooperativity model.