Tränkle C, Mies-Klomfass E, Cid M H, Holzgrabe U, Mohr K
Department of Pharmacology and Toxicology, Institute of Pharmacy, University of Bonn, An der Immenburg 4, D-53121 Bonn, Germany.
Mol Pharmacol. 1998 Jul;54(1):139-45. doi: 10.1124/mol.54.1.139.
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 degrees) in the presence of 1 microM NMS. Homologous competition experiments revealed two components of saturable radioligand binding: one with a high affinity (KD = 2 nM) and small capacity ( approximately 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.
毒蕈碱型乙酰胆碱受体在一个与传统配体所使用的正构位点不同的位点结合变构调节剂。我们在心脏组织中测试了调节剂与配体占据的毒蕈碱M2受体的结合是否是一个可以使用放射性变构剂检测到的优先事件。新合成的二甲基-W84（N，N'-双[3-（1，3-二氢-1，3-二氧代-4-甲基-2H-异吲哚-2-基）丙基]-N，N，N'，N'-四甲基-1，6-己二铵二碘化物）对被传统拮抗剂N-甲基东莨菪碱（NMS）占据的M2受体具有特别高的效力；在3 nM的EC50，diss值下，[3H]NMS的解离被最大程度地延迟了一半。使用双复磷作为“变构拮抗剂”，发现了二甲基-W84与假定的共同变构位点相互作用的证据。在1μM NMS存在下，在猪心脏匀浆（4 mM Na2HPO4，1 mM KH2PO4，pH 7.4，23℃）中测量[3H]二甲基-W84（0.3 nM；比活，168 Ci/mmol）的结合。同源竞争实验揭示了可饱和放射性配体结合的两个成分：一个具有高亲和力（KD = 2 nM）和小容量（约占总可饱和结合的30%），另一个亲和力低20,000倍。高亲和力位点的Bmax值（68 fmol/mg蛋白质）与通过[3H]NMS测定的毒蕈碱受体密度（79 fmol/mg）相匹配。原型变构剂阿库氯铵、W84（放射性配体母体化合物）和加拉明浓度依赖性地取代高亲和力[3H]二甲基-W84结合（pKi值分别为8.62、7.83和6.72）。调节剂的结合亲和力与其变构稳定NMS/受体复合物的效力（EC50，diss分别为8.40、7.72和6.74）具有极好的相关性。我们得出结论，[3H]二甲基-W84的高亲和力结合反映了M2受体共同变构位点的占据。
