Abstract
Muscarinic acetylcholine receptors bind allosteric modulators at a site apart from the orthosteric site used by conventional ligands. We tested in cardiac tissue whether modulator binding to ligand-occupied muscarinic M2 receptors is a preferential event that can be detected using a radioactive allosteric agent. The newly synthesized 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) has a particular high potency at M2 receptors occupied by the conventional antagonistN-methylscopolamine (NMS); dissociation of [3H]NMS is half-maximally retarded at an EC50,diss value of 3 nm. Using obidoxime as an “allosteric antagonist,” evidence was found that dimethyl-W84 interacts with the postulated common allosteric site. Binding of [3H]dimethyl-W84 (0.3 nm; specific activity, 168 Ci/mmol) was measured in porcine heart homogenates (4 mm Na2HPO4, 1 mmKH2PO4, pH 7.4, 23°) in the presence of 1 μm NMS. Homologous competition experiments revealed two components of saturable radioligand binding: one with a high affinity (K D = 2 nm) and small capacity (≈30% of total saturable binding) and the other with a 20,000-fold lower affinity. The B max value of the high affinity sites (68 fmol/mg protein) matched muscarinic receptor density as determined by [3H]NMS (79 fmol/mg). Prototype allosteric agents, alcuronium, W84 (the parent compound of the radioligand), and gallamine, displaced high affinity [3H]dimethyl-W84 binding concentration-dependently (pK i values = 8.62, 7.83, and 6.72, respectively). The binding affinities of the modulators were in excellent correlation with their potencies to allosterically stabilize NMS/receptor complexes (EC50,diss = 8.40, 7.72, and 6.74, respectively). We conclude that high affinity binding of [3H]dimethyl-W84 reflects occupation of the common allosteric site of M2 receptors.
Footnotes
- Received December 8, 1997.
- Accepted March 12, 1998.
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Send reprint requests to: Klaus Mohr, Department of Pharmacology & 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 und the Fonds der Chemischen Industrie, Germany. H.M.B.C. acknowledges a grant given by the Katholischer Akademischer Austauschdienst.
- The American Society for Pharmacology and Experimental Therapeutics
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