Impermeant potential-sensitive oxonol dyes. I: Evidence for an on-off mechanism

This series of papers addresses the mechanism by which certain impermeant oxonol dyes respond to membrane-potential changes, denoted delta Em. Hemispherical oxidized cholesterol bilayer membranes provided a controlled model membrane system for determining the dependence of the light absorption signal from the dye on parameters such as the wavelength and polarization of the light illuminating the membrane, the structure of the dye, and delta Em. This paper is concerned with the determination and analysis of absorption spectral changes of the dye RGA461 during trains of step changes of Em. The wavelength dependence of the absorption signal is consistent with an "on-off" mechanism in which dye molecules are driven by potential changes between an aqueous region just off the membrane and a relatively nonpolar binding site on the membrane. Polarization data indicate that dye molecules in the membrane site tend to orient with the long axis of the chromophore perpendicular to the surface of the membrane. Experiments with hyperpolarized human red blood cells confirmed that the impermeant oxonols undergo a potential-dependent partition between the membrane and the bathing medium.