RT Journal Article SR Electronic T1 Identification of a [3H]Ligand for the Common Allosteric Site of Muscarinic Acetylcholine M2 Receptors JF Molecular Pharmacology JO Mol Pharmacol FD American Society for Pharmacology and Experimental Therapeutics SP 139 OP 145 DO 10.1124/mol.54.1.139 VO 54 IS 1 A1 Christian Tränkle A1 Elisabeth Mies-Klomfass A1 Mario H. Botero Cid A1 Ulrike Holzgrabe A1 Klaus Mohr YR 1998 UL http://molpharm.aspetjournals.org/content/54/1/139.abstract AB 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.