Direct spectrophotometric detection of cation flux in membrane vesicles: Stopped-flow measurements of acetylcholine-receptor-mediated ion flux

Abstract

The development of a spectrophotometric stopped-flow method to measure ion flux in membrane vesicles in the millisecond to minute time region is described in detail. The technique is based on fluorescence quenching of an entrapped fluorophore (anthracene-1,5-disulfonic acid) by Cs +. The method has been applied to the measurement of acetylcholine-receptor-mediated ion flux in membrane vesicles prepared from the electric organs of both Electrophorus electricus and Torpedo californica. The method is applicable to any vesicle system in which Cs + can substitute for either Na + or K +. Loading of vesicles with the fluorescent dye is accomplished using the routine procedure for making the vesicles. The dye-loaded vesicles can be stored in liquid nitrogen before use. Neither the dye-loading procedure nor the presence of Cs + changes the permeability of the membrane to ions, allowing ion-translocation measurements to be made in the millisecond to minute time region. The stopped-flow design presented allows two sequential mixings of solutions. The relationship between fluorescence quenching and ion flux as well as the interpretation of the ion flux data is described. It is shown that the data obtained with stopped-flow and Cs + is identical to data obtained previously using a quench-flow technique and 86Rb +. The advantages of the present method over the quench-flow technique and a similar stopped-flow technique developed previously based on T1 + are described in detail.