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 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 [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 (EC_50,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.