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Research ArticleArticle

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
Molecular Pharmacology November 1999, 56 (5) 962-965; DOI: https://doi.org/10.1124/mol.56.5.962
Christian Tränkle
Department of Pharmacology and Toxicology, Institute of Pharmacy, University of Bonn, Bonn, Germany
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Oliver Weyand
Department of Pharmacology and Toxicology, Institute of Pharmacy, University of Bonn, Bonn, Germany
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Alexandra Schröter
Department of Pharmacology and Toxicology, Institute of Pharmacy, University of Bonn, Bonn, Germany
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Klaus Mohr
Department of Pharmacology and Toxicology, Institute of Pharmacy, University of Bonn, Bonn, Germany
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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.
  • 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

  • This work was supported by the Deutsche Forschungsgemeinschaft.

  • The American Society for Pharmacology and Experimental Therapeutics
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Molecular Pharmacology: 56 (5)
Molecular Pharmacology
Vol. 56, Issue 5
1 Nov 1999
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Research ArticleArticle

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
Molecular Pharmacology November 1, 1999, 56 (5) 962-965; DOI: https://doi.org/10.1124/mol.56.5.962

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Research ArticleArticle

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
Molecular Pharmacology November 1, 1999, 56 (5) 962-965; DOI: https://doi.org/10.1124/mol.56.5.962
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