C. Trankle et al., IDENTIFICATION OF A [H-3]LIGAND FOR THE COMMON ALLOSTERIC SITE OF MUSCARINIC ACETYLCHOLINE M-2 RECEPTORS, Molecular pharmacology, 54(1), 1998, pp. 139-145
Muscarinic acetylcholine receptors bind allosteric modulators at a sit
e apart from the orthosteric site used by conventional ligands. We tes
ted in cardiac tissue whether modulator binding to ligand-occupied mus
carinic M-2 receptors is a preferential event that can be detected usi
ng a radioactive allosteric agent. The newly synthesized dimethyl-W84
)propyl]-N,N,N',N'-tetramethyl-1,6-hexanediaminium diiodide) has a par
ticular high potency at M-2 receptors occupied by the conventional ant
agonist N-methylscopolamine (NMS); dissociation of [H-3]NMS is half-ma
ximally retarded at an EC50,diss value of 3 nM. Using obidoxime as an
''allosteric antagonist,'' evidence was found that dimethyl-W84 intera
cts with the postulated common allosteric site. Binding of [H-3]dimeth
yl-W84 (0.3 nM; specific activity, 168 Ci/mmol) was measured in porcin
e heart homogenates (4 mM Na2HPO4, 1 mM KH2PO4, pH 7.4, 23 degrees) in
the presence of 1 mu M NMS. Homologous competition experiments reveal
ed two components of saturable radioligand binding: one with a high af
finity (K-D = 2 nM) and small capacity (approximate to 30% of total sa
turable 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 mu
scarinic receptor density as determined by [H-3]NMS (79 fmol/mg). Prot
otype allosteric agents, alcuronium, W84 (the parent compound of the r
adioligand), and gallamine, displaced high affinity [H-3]dimethyl-W84
binding concentration-dependently (pK(i) values = 8.62, 7.83, and 6.72
, respectively). The binding affinities of the modulators were in exce
llent 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 [H-3]dimethyl-W84 reflects o
ccupation of the common allosteric site of M-2 receptors.