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 ofcation 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.

A Thallium-Sensitive, Fluorescence-Based Assay for Detecting and Characterizing Potassium Channel Modulators in Mammalian Cells

“…The permeability of Tl + through K + channels is well known, 8,9 and assays based on the Tl + -sensitive fluors have even been described. [10][11][12][13][14] However, the fluors and the assays developed from them have not been widely adopted because they have low utility due to the spectral properties of the dyes, small signals, and the poor solubility of Tl + in chloride (Cl -)-containing buffer systems. 14 The present study describes a Tl + -sensitive fluorescent dye and a buffer system that largely overcome the deficiencies of prior techniques, yielding an assay system capable of allowing rapid, facile, accurate, and precise measurements of K + channel activity suitable for screening large chemical libraries.…”

Ion Channels in Liposomes

“…The system behaves as though at low agonist concentra tions, most of the receptors desensitize before opening, while the reverse is true tion process, however, could be easily followed in the quench-flow system, and was found to agree well with the corresponding behavior in the native Torpedo membrane vesicles (44). Wu et al (46) increased the time resolution to about 8 ms by monitoring Tl + influx via a fluorescence-quenching technique.…”

Stopped-Flow Fluorometric Ion Flux Assay for Ligand-Gated Ion Channel Studies

“…If conditions favor less channel activity, the Tl + flux will be comparably slower, as will the fluorescence quenching rate (Figure 1b). This assay format was first used for acetylcholine receptors [5,6] and fragmented sarcoplasmic reticulum membrane preparations [7,8]. A variant, based on Tl + selective fluorescent indicators, later was employed and commercialized for cell-based flux assays [9].…”