IDENTIFICATION OF A [H-3]LIGAND FOR THE COMMON ALLOSTERIC SITE OF MUSCARINIC ACETYLCHOLINE M-2 RECEPTORS

Citation
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
Citations number
26
Categorie Soggetti
Pharmacology & Pharmacy",Biology
Journal title
ISSN journal
0026895X
Volume
54
Issue
1
Year of publication
1998
Pages
139 - 145
Database
ISI
SICI code
0026-895X(1998)54:1<139:IOA[FT>2.0.ZU;2-W
Abstract
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.