Walker J W, Takeyasu K, McNamee M G
Biochemistry. 1982 Oct 26;21(22):5384-9. doi: 10.1021/bi00265a001.
By use of a quench-flow technique to measure tracer ion flux rates in a physiologically significant time domain, the kinetics of activation and inactivation of purified reconstituted acetylcholine receptor (AChR) were investigated. After solubilization in sodium cholate, purification by affinity chromatography, and reconstitution into soybean lipids, the AChR from Torpedo californica displayed a characteristically fast rate of ion influx measured with 86Rb+. At 4 degrees C 1 mM carbamoylcholine (Carb) stimulated a fast (t1/2 = 7 ms) first-order filling of vesicle internal volume that presented a 10(4)-fold stimulation of ion flux rate by Carb. The concentration dependence of activation was sigmoidal with a half-maximal value at 3 X 10(-4) M Carb. In the presence of Carb, the purified AChR also underwent a two-step inactivation (desensitization) process. Inactivation was measured by preincubating AChR with Carb for various times (milliseconds to minutes) and then measuring the 86Rb+ influx rate. The two inactivation processes were each characterized by a distinct maximum rate (5.3 and 0.10 s-1) and by a different dependence on Carb concentration. The slow phase of inactivation gave a half-maximal rate at 2.5 X 10(-4) M Carb, and the fast inactivation was half-maximal at 1.3 X 10(-3) M Carb. The concentration dependence curves for both inactivation processes were approximately hyperbolic. The results are discussed in terms of models that describe the relationship between ligand binding and the processes of channel activation and desensitization.
通过使用淬灭流动技术在生理相关的时间域中测量示踪离子通量率，研究了纯化的重组乙酰胆碱受体（AChR）的激活和失活动力学。在胆酸钠中溶解、通过亲和色谱纯化并重构到大豆脂质中后，来自加州电鳐的AChR显示出用86Rb+测量的特征性快速离子内流速率。在4℃下，1 mM氨甲酰胆碱（Carb）刺激了囊泡内部体积的快速（t1/2 = 7 ms）一级填充，这使得Carb对离子通量率产生了10^4倍的刺激。激活的浓度依赖性呈S形，在3×10^(-4) M Carb时达到半最大值。在Carb存在下，纯化的AChR还经历了两步失活（脱敏）过程。通过将AChR与Carb预孵育不同时间（毫秒到分钟），然后测量86Rb+内流速率来测量失活。这两个失活过程各自具有不同的最大速率（5.3和0.10 s^(-1)）以及对Carb浓度的不同依赖性。失活的慢相在2.5×10^(-4) M Carb时达到半最大速率，快速失活在1.3×10^(-3) M Carb时达到半最大速率。两个失活过程的浓度依赖性曲线近似为双曲线。根据描述配体结合与通道激活和脱敏过程之间关系的模型对结果进行了讨论。
