Optical detection of membrane dipole potential: avoidance of fluidity and dye-induced effects

Fluorescent styrylpyridinium dyes have recently been suggested as probes of the membrane dipole potential and of the kinetics of electrogenic ion pumps. It is necessary, however, to be able to confidently attribute observed fluorescence changes to electrical effects alone and avoid interference from changes in membrane fluidity. Furthermore, the effect of the dyes themselves on the dipole potential must be investigated. The effect of membrane fluidity on the fluorescence excitation and emission spectra of the dyes RH421 and di-8-ANEPPS have been investigated in lipid vesicles by temperature scans between 15 and 60 degrees C. Both dyes show significant temperature-dependent shifts of their excitation spectra, the magnitude of which depend on the emission wavelength and on the lipid structure. In order to eliminate membrane fluidity effects, fluorescence must be detected at the red edge of the emission spectrum; in this case 670 nm. In order to avoid dye-induced shifts of the excitation spectra of membrane-bound dye, an excess molar ratio of lipid to dye of at least 200-fold is necessary. Fluorescence ratio measurements indicate qualitatively that dimyristoylphosphatidylcholine has a significantly higher dipole potential than that of dioleoylphosphatidylcholine.