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Identification of a [ 3 H]Ligand for the Common Allosteric Site of Muscarinic Acetylcholine M 2 Receptors

1998; American Society for Pharmacology and Experimental Therapeutics; Volume: 54; Issue: 1 Linguagem: Inglês

10.1124/mol.54.1.139

1521-0111

Christian Tränkle, Elisabeth Mies-Klomfass, Mario H. Botero Cid, Ulrike Holzgrabe, Klaus Mohr,

Nicotinic Acetylcholine Receptors Study

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 M 2 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-2 H -isoindol-2-yl)propyl]- N , N , N ′, N ′-tetramethyl-1,6-hexanediaminium diiodide) has a particular high potency at M 2 receptors occupied by the conventional antagonist N -methylscopolamine (NMS); dissociation of [ 3 H]NMS is half-maximally retarded at an EC 50,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 [ 3 H]dimethyl-W84 (0.3 nm; specific activity, 168 Ci/mmol) was measured in porcine heart homogenates (4 mm Na 2 HPO 4 , 1 mmKH 2 PO 4 , 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 [ 3 H]NMS (79 fmol/mg). Prototype allosteric agents, alcuronium, W84 (the parent compound of the radioligand), and gallamine, displaced high affinity [ 3 H]dimethyl-W84 binding concentration-dependently (p K 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 (EC 50,diss = 8.40, 7.72, and 6.74, respectively). We conclude that high affinity binding of [ 3 H]dimethyl-W84 reflects occupation of the common allosteric site of M 2 receptors.