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Vol. 54, Issue 1, 139-145, July 1998
Department of Pharmacology and Toxicology (C.T., E.M.-K., K.M.),
Institute of Pharmacy, University of Bonn, An der Immenburg 4, D-53121 Bonn, Germany, and
Department of Pharmaceutical
Chemistry (M.H.B.C., U.H.), Institute of Pharmacy, University of Bonn,
Kreuzbergweg 26, D-53115 Bonn, Germany
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°) in the presence of 1 µM NMS. Homologous competition experiments revealed two
components of saturable radioligand binding: one with a high affinity
(KD = 2 nM) and
small capacity (
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.
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