Quantitation of cation transport by reconstituted membrane vesicles containing purified acetylcholine receptor

Abstract

A stopped-flow spectroscopic technique was used to study the kinetics of ion transport by reconstituted membrane preparations containing purified acetylcholine receptor. Influx of thallium (I) into membrane vesicles was monitored as a decrease, due to quenching by the thallous ion, in the fluorescence of an entrapped fluorophore. In a reproducible manner, the reconstituted receptor responded to cholinergic agonists by mediating rapid ion transport in the millisecond time range. The effect of agonists was blocked by receptor desensitization and by histrionicotoxin and was absent in membrane vesicles lacking receptor. Analysis of the fast kinetics of cation transport produced by saturating concentrations of agonists yielded an estimated rate of transport through a single reconstituted receptor channel. Comparison of this rate with those reported for in vivo preparations and for purified membranes shows that the reconstituted protein closely resembles the physiologically active receptor.