The research proposed is aimed at characterizing quantitatively the dependence of the spectral properties of a number of """"""""voltage-sensing"""""""" dyes on physical variables, such as transmembrane and electrostatic potentials, dielectric constant, and fluidity, associated with membranes. In addition, experiments are proposed to determine whether and how these dyes alter the properties of the membranes with which they are associated. These analyses are based on electrical and spectroscopic studies of lipid bilayer membranes, for which variations in lipid composition and aqueous ion compositions can be readily made, as well as excitable cell membranes. The importance of these studies lies in the fact that the spectral responses of these dyes, while potentially invaluable indicators of electrical processes occurring in membranes of excitable cells, such as heart muscle and nerve, are also sensitive to a variety of other membrane variables. Thus the same change in transmembrane potential in two different cell types yields different changes in a given dye's spectrum. These dyes are also being investigated as useful indicators of oncogenic transformation in hemopoietic tissue since, for example, they yield different spectral responses in blood cells from leukemic patients than from controls. The membrane variable(s) responsible for these apparent differences, and the mechanisms by which the dyes report them, are not understood, however. The types of analyses proposed in the present project should provide insight into how dyes respond to different membrane properties and what dye structures are best for sensing specific membrane variables as well as leading to a method for """"""""calibrating"""""""" the spectral responses of these dyes so that they can be used as quantitative probes of such membrane variables as transmembrane potential, electrostatic potential, fluidity, and dielectric constant.
