Prince R J, Sine S M
Receptor Biology Laboratory, Department of Physiology and Biophysics, Mayo Foundation, Rochester, Minnesota 55905, USA.
J Biol Chem. 1998 Apr 3;273(14):7843-9. doi: 10.1074/jbc.273.14.7843.
Ligand binding sites in fetal (alpha2betagammadelta) and adult (alpha2betadeltaepsilon) muscle acetylcholine receptors are formed by alphadelta, alphagamma, or alphaepsilon subunit pairs. Each type of binding site shows unique ligand selectivity due to different contributions by the delta, gamma, or epsilon subunits. The present study compares epibatidine and carbamylcholine binding in terms of their site and state selectivities for muscle receptors expressed in human embryonic kidney 293 cells. Measurements of binding to alphagamma, alphadelta, and alphaepsilon intracellular complexes reveal opposite site selectivities between epibatidine and carbamylcholine; for epibatidine the rank order of affinities is alphaepsilon > alphagamma > alphadelta, whereas for carbamylcholine the rank order is alphadelta congruent with alphaepsilon > alphagamma. Because the relative affinities of intracellular complexes resemble those of receptors in the closed activable state, the results suggest that epibatidine binds with unique site selectivity in activating the muscle receptor. Measurements of binding at equilibrium show that both enantiomers of epibatidine bind to adult and fetal receptors with shallow but monophasic binding curves. However, when receptors are fully desensitized, epibatidine binds in a biphasic manner, with dissociation constants of the two components differing by more than 170-fold. Studies of subunit-omitted receptors (alpha2betadelta2, alpha2betagamma2, and alpha2betaepsilon2) reveal that in the desensitized state, the alphadelta interface forms the low affinity epibatidine site, whereas the alphagamma and alphaepsilon interfaces form high affinity sites. In contrast to epibatidine, carbamylcholine shows little site selectivity for desensitized fetal or adult receptors. Thus epibatidine is a potentially valuable probe of acetylcholine receptor binding site structure and of elements that confer state-dependent selectivities of the binding sites.
胎儿（α2βγδ）和成体（α2βδε）肌肉乙酰胆碱受体中的配体结合位点由αδ、αγ或αε亚基对形成。由于δ、γ或ε亚基的不同贡献，每种类型的结合位点都表现出独特的配体选择性。本研究比较了埃博霉素和氨甲酰胆碱在人胚肾293细胞中表达的肌肉受体的位点和状态选择性方面的结合情况。对αγ、αδ和αε细胞内复合物结合的测量揭示了埃博霉素和氨甲酰胆碱之间相反的位点选择性；对于埃博霉素，亲和力的顺序是αε＞αγ＞αδ，而对于氨甲酰胆碱，顺序是αδ≡αε＞αγ。因为细胞内复合物的相对亲和力类似于处于关闭可激活状态的受体的亲和力，结果表明埃博霉素在激活肌肉受体时以独特的位点选择性结合。平衡结合测量表明，埃博霉素的两种对映体都以浅但单相的结合曲线与成体和胎儿受体结合。然而，当受体完全脱敏时，埃博霉素以双相方式结合，两种成分的解离常数相差超过170倍。对缺失亚基的受体（α2βδ2、α2βγ2和α2βε2）的研究表明，在脱敏状态下，αδ界面形成低亲和力的埃博霉素位点，而αγ和αε界面形成高亲和力位点。与埃博霉素相反，氨甲酰胆碱对脱敏的胎儿或成体受体几乎没有位点选择性。因此，埃博霉素是乙酰胆碱受体结合位点结构以及赋予结合位点状态依赖性选择性的元件的潜在有价值的探针。
