Walker J W, Richardson C A, McNamee M G
Biochemistry. 1984 May 22;23(11):2329-38. doi: 10.1021/bi00306a002.
The effects of thio-group modifications on the ion permeability control and ligand binding properties of the acetylcholine receptor were measured in reconstituted membranes prepared from purified Torpedo californica acetylcholine receptor and soybean lipids (asolectin). A quench flow device was used to obtain subsecond time resolution for agonist-stimulated cation influx using carbamylcholine chloride (Carb) as the ligand and 86Rb+ as the cation. The effects of disulfide reduction with dithiothreitol (DTT), affinity alkylation with [4-(N-maleimido)benzyl]trimethylammonium ion and bromoacetylcholine, and nonspecific alkylation with N-ethylmaleimide and N-benzylmaleimide were examined. Activation, fast inactivation, and slow inactivation rates were measured on the chemically modified membranes. The flux results were compared with similar measurements on native membranes, and the role of vesicle size, heterogeneity, and influx time on ion flux results was analyzed. Major conclusions are that the binding sites that react with affinity labels are the same sites that mediate ligand-activated ion flux and that blockade of one of the two ligand binding sites is sufficient to block about 95% of the ion flux response. The main effect of DTT reduction is to shift the EC50 values for activation and slow inactivation to higher Carb concentrations, consistent with a decrease in binding affinity for Carb. The EC50 value for fast inactivation was not affected by DTT. However, the maximum rate of ion flux activation and the maximum rate of fast inactivation were decreased 2-fold after DTT treatment.
在由纯化的加州电鳐乙酰胆碱受体和大豆脂质（大豆卵磷脂）制备的重构膜中，测定了硫基修饰对乙酰胆碱受体离子通透性控制和配体结合特性的影响。使用淬灭流动装置，以氯化氨甲酰胆碱（Carb）作为配体、86Rb⁺作为阳离子，获得激动剂刺激的阳离子内流的亚秒级时间分辨率。研究了用二硫苏糖醇（DTT）进行二硫键还原、用[4-(N-马来酰亚胺基)苄基]三甲基铵离子和溴乙酰胆碱进行亲和烷基化以及用N-乙基马来酰亚胺和N-苄基马来酰亚胺进行非特异性烷基化的效果。在化学修饰的膜上测量了激活、快速失活和缓慢失活速率。将通量结果与天然膜上的类似测量结果进行比较，并分析了囊泡大小、异质性和内流时间对离子通量结果的作用。主要结论是，与亲和标记反应的结合位点是介导配体激活离子通量的相同位点，并且两个配体结合位点之一的阻断足以阻断约95%的离子通量反应。DTT还原的主要作用是将激活和缓慢失活的EC50值转移到更高的Carb浓度，这与对Carb的结合亲和力降低一致。快速失活的EC50值不受DTT影响。然而，DTT处理后离子通量激活的最大速率和快速失活的最大速率降低了2倍。
