title: Identification of a [3H]Ligand for the common allosteric site of muscarinic acetylcholine M2 receptors.
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 antagonist N-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 mM KH2PO4, pH 7.4, 23 degrees) in the presence of 1 microM NMS. Homologous competition experiments revealed two components of saturable radioligand binding: one with a high affinity (KD = 2 nM) and small capacity ( approximately 30% of total saturable binding) and the other with a 20,000-fold lower affinity. The Bmax 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 (pKi 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.
authors.affiliation: Department of Pharmacology and Toxicology, Institute of Pharmacy, University of Bonn, An der Immenburg 4, D-53121 Bonn, Germany.
authors.forename: C
authors.initials: C
authors.lastname: Tränkle
authors.forename: E
authors.initials: E
authors.lastname: Mies-Klomfass
authors.forename: M H
authors.initials: MH
authors.lastname: Cid
authors.forename: U
authors.initials: U
authors.lastname: Holzgrabe
authors.forename: K
authors.initials: K
authors.lastname: Mohr
mesh_terms: D000495:Allosteric Site / Q000187:drug effects*; D000818:Animals; D002801:Cholinesterase Reactivators / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004347:Drug Interactions; D006321:Heart / Q000187:drug effects; D054833:Isoindoles; D008024:Ligands; D009206:Myocardium / Q000378:metabolism; D019832:N-Methylscopolamine / Q000494:pharmacology; D009768:Obidoxime Chloride / Q000494:pharmacology; D010276:Parasympatholytics / Q000494:pharmacology; D010797:Phthalimides / Q000138:chemical synthesis / Q000737:chemistry / Q000494:pharmacology*; D019275:Radiopharmaceuticals / Q000494:pharmacology; D043585:Receptor, Muscarinic M2; D011976:Receptors, Muscarinic / Q000187:drug effects* / Q000378:metabolism; D013552:Swine
chemical_list: D002801:Cholinesterase Reactivators; D054833:Isoindoles; D008024:Ligands; D010276:Parasympatholytics; D010797:Phthalimides; D019275:Radiopharmaceuticals; D043585:Receptor, Muscarinic M2; D011976:Receptors, Muscarinic; C113497:dimethyl-W84; D009768:Obidoxime Chloride; D019832:N-Methylscopolamine