Activation and inactivation kinetics of Torpedo californica acetylcholine receptor in reconstituted membranes

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Abstract
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 T. californica displayed a characteristically fast rate of ion influx measured with 86Rb+. At 4.degree. C 1 mM carbamoylcholine (Carb) stimulated a fast (t1/2 [half-time] = 7 ms) 1st-order filling of vesicle internal volume that represented a 104-fold stimulation of ion flux rate by Carb. The concentration dependence of activation was sigmoidal with a half-maximal value at 3 .times. 10-4 M Carb. In the presence of Carb, the purified AChR also underwent a 2-step inactivation (desensitization) process. Inactivation was measured by preincubating AChR with Carb for various times (ms-min) and then measuring the 86Rb+ influx rate. The 2 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 .times. 10-4 M Carb, and the fast inactivation was half-maximal at 1.3 .times. 10-3 M Carb. The concentration dependence curves for both inactivation processes were approximately hyperbolic. Models that describe the relationship between ligand binding and the processes of channel activation and desensitization were discussed.