Impermeant potential-sensitive oxonol dyes: I. Evidence for an “On-off” mechanism

Abstract

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.

Volume-Sensitive Chloride Channels Do Not Mediate Activation-Induced Chloride Efflux in Human Neutrophils

“…Unlike diBA-(5)-C 4 , WW781 is membrane impermeable (25), and therefore its effects are expected to be restricted to sites accessible from the extracellular side of the membrane. In our experiments this was confirmed by the absence of blue color inside the cells.…”

“…3 and 4). Because the dark blue WW781 does not cause any noticeable staining of the cells, it appears that it remains extracellular, probably because of its very polar sulfonic acid group (25), and thus inhibits by binding to a site that is accessible from the extracellular side of the membrane. This is consistent with the very small voltage dependence of inhibition, which suggests that the doubly negatively charged WW781 binds to a site that is readily accessible from the outside.…”

Impermeant potential-sensitive oxonol dyes: II. The dependence of the absorption signal on the length of alkyl substituents attached to the dye

“…The oxidized cholesterol bilayer lipid membrane system for detecting optical signals is described in the previous paper (George et al (1988a). Changes (AI) in the intensity of quasimonochromatic light passing through the bottom of the oxidized cholesterol hemispherical bilayer membrane (apparatus details in George et al, 1988a) were measured during a train of 5-msec voltage steps that alternated between -50 and +50 mV.…”

“…2 and in the Table were Table measured at the wavelength where the maximum signal was obtained (630 nm for the barbituric acid derivatives and 660 nm for the thiobarbituric acid analogs). Absorption spectra were obtained as described by George et al (1988a).…”

“…First, the membranes are relatively stable in the presence of a large number of different dye analogs. Second, the spherical configuration of the oxidized cholesterol membrane developed by Dragsten and Webb (1978) provided an excellent geometry for the polarization studies of the first paper in this series (George et al, 1988a). In the present study, we have measured the potential-dependent absorption change AA of 43 analogs of WW781 associated with oxidized cholesterol bilayer lipid membranes.…”