Independent activation of the acetylcholine receptor from Torpedo californica at two sites.
Independent activation of the acetylcholine receptor from Torpedo californica at two sites.
复制标题
加州鱼雷乙酰胆碱受体在两个位点的独立激活。
DOI：
10.1021/bi00546a010
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发表时间：
1980
期刊：
影响因子：
2.9
通讯作者：
M. McNamee
中科院分区：
文献类型：
--
作者：
A. Delegeane;M. McNamee
从加利福尼亚电鳐（Torpedo californica）的电沉积组织中制备了富含乙酰胆碱受体的膜泡。用二硫苏糖醇还原受体以暴露配体结合位点附近的巯基，然后以下列方式之一处理：（1）以下列方式之一亲和烷基化处理：（1）用受体激活剂溴乙酰胆碱亲合烷基化，（2）用受体抑制剂马来酰亚胺苄基三甲基铵亲合烷基化，或（3）用二硫代双（2-硝基苯甲酸酯）再氧化至天然状态。亲和标记物阻断了α-银环蛇毒素的一半结合位点。基于毒素结合动力学的研究，未被亲和标记保护的毒素位点被氨基甲酰胆碱保护。通过钠离子通量程序测量天然和亲和烷基化受体的功能反应。在没有添加胆碱能激活剂的情况下，仅观察到缓慢的离子通量。在受体激活剂氨甲酰胆碱的存在下，天然和修饰的膜都显示出与功能性受体相关的钠通量增加。50%最大响应所需的氨基甲酰胆碱的浓度在亲和标记的膜中更高。预孵育的膜与氨甲酰胆碱阻断增加的离子通量，表明可以诱导脱敏。结果为乙酰胆碱受体上存在两个功能位点提供了证据。每个位点对应于银环蛇毒素结合位点，并且可以独立地被激活和脱敏。
Membrane vesicles enriched in acetylcholine receptor were prepared from the electroplax tissue of Torpedo californica. The receptor was reduced with dithiothreitol to expose a sulfhydryl group near the ligand binidng site and then treated in one of the following ways: (1) affinity alkylated treated in one of the following ways: (1) affinity alkylated with bromoacetylcholine, a receptor activator, (2) affinity alkylated with maleimidobenzyltrimethylammonium, a receptor inhibitor, or (3) reoxidized to the native state with dithiobis(2-nitrobenzoate). The affinity labels blocked half of the binding sites for alpha-bungarotoxin. The toxin sites not protected by the affinity labels were protected by carbamylcholine based on studies of toxin binding kinetics. The functional response of native and affinity-alkylated receptors was measured by a sodium ion flux procedure. In the absence of added cholinergic activators, only slow ion flux was observed. In the presence of carbamylcholine, a receptor activator, both native and modified membranes showed the increased sodium flux associated with functional receptors. The concentration of carbamylcholine required for a 50% maximal response was higher in the affinity-labeled membranes. Preincubation of the membranes with carbamylcholine blocked the increased ion flux, indicating that desensitization could be induced. The results provide evidence for the existence of two functional sites on the acetylcholine receptor. Each site corresponds to a bungarotoxin binding site and can be independently activated and desensitized.