Kinetics of Potential-Sensitive Fluorescent Dye Interaction with Lipid Vesicles and Application to the Measurement of Membrane Potential Transients Generated by the Na,K-Pump

Abstract

The interaction of the dyes oxonol V and oxonol VI with unilamellar lipid vesicles was investigated using a fluorescence stopped-flow technique. On mixing with the vesicles, both dyes exhibit a biphasic increase in their fluorescence. Based on the dependence of the rates on vesicle concentration, the two phases have been interpreted as being due to a rapid binding of dye to the lipid membrane followed by a slower diffusion of dye across the membrane and binding to the internal monolayer. The response times of the dyes to a rapid jump in the membrane potential has also been investigated. Oxonol VI was found to respond to the potential change in less than one second, whereas oxonol V required several minutes. This has been attributed to a lower mobility of oxonol V within the lipid membrane. The application of oxonol VI to the kinetic measurement of the potential generated by the membrane protein Na,K-ATPase, reconstituted into lipid vesicles, is shown.