Lee N H, el-Fakahany E E
Department of Pharmacology and Toxicology, University of Maryland School of Pharmacy, Baltimore.
J Pharmacol Exp Ther. 1988 Sep;246(3):829-38.
The binding profile of the positively charged muscarinic antagonist, gallamine, was studied in rat heart homogenates. A proportion of the binding sites labeled by the tertiary muscarinic ligands [( 3H]quinuclidinyl benzilate (QNB) and [3H]atropine) were inaccessible to their quaternary analogs [( 3H]N-methyl-QNB (NMeQNB) and [3H]-N-methylscopolamine (NMS)] or gallamine. Whereas gallamine displaced the binding of [3H]NMeQNB with high affinity, biphasic competition curves were observed using [3H]NMS only at higher ligand concentrations. The rank order of potency of gallamine in allosterically decelerating ligand dissociation kinetics was: [3H]NMS greater than [3H]atropine greater than [3H]NMeQNB greater than [3H]QNB. Our calculations demonstrate that the displayed heterogeneity of gallamine binding sites detected using [3H]NMS, but not the tertiary ligands, might be accounted for by the allosteric modification of the binding of this ligand by gallamine. Based on these findings, the exhibited binding profile of gallamine to muscarinic receptors is influenced strongly by ligand choice, and also by the ligand concentration used in the binding experiment. Furthermore, it is concluded that gallamine binds to three major sites on the muscarinic receptor, thereby revealing an apparent heterogeneity of its binding sites, even in a tissue which presumably possesses one major muscarinic receptor subtype such as the heart. According to several lines of evidence, gallamine binds competitively and with high affinity to NMS-accessible sites on the receptor. Under certain experimental conditions, it also appears to identify another low-affinity site, either due to its binding to NMS-inaccessible sites or through its differential ability to alter the binding of ligands to the main binding domain on the receptor in an allosteric fashion.
在大鼠心脏匀浆中研究了带正电荷的毒蕈碱拮抗剂加拉明的结合特性。叔胺类毒蕈碱配体[（3H）喹核醇基苯甲酸酯（QNB）和（3H）阿托品]标记的一部分结合位点，其季铵类似物[（3H）N-甲基-QNB（NMeQNB）和（3H）-N-甲基东莨菪碱（NMS）]或加拉明无法接近。虽然加拉明以高亲和力取代（3H）NMeQNB的结合，但仅在较高配体浓度下使用（3H）NMS时观察到双相竞争曲线。加拉明在变构减速配体解离动力学中的效力顺序为：（3H）NMS＞（3H）阿托品＞（3H）NMeQNB＞（3H）QNB。我们的计算表明，使用（3H）NMS而非叔胺类配体检测到的加拉明结合位点的异质性，可能是由于加拉明对该配体结合的变构修饰所致。基于这些发现，加拉明与毒蕈碱受体的结合特性受到配体选择以及结合实验中所用配体浓度的强烈影响。此外，得出的结论是，加拉明与毒蕈碱受体上的三个主要位点结合，从而揭示了其结合位点的明显异质性，即使在一个可能仅拥有一种主要毒蕈碱受体亚型的组织如心脏中也是如此。根据多条证据，加拉明与受体上NMS可及位点竞争性且高亲和力结合。在某些实验条件下，它似乎还能识别另一个低亲和力位点，这要么是由于它与NMS不可及位点结合，要么是由于它以变构方式改变配体与受体主要结合域结合的不同能力。
