The main objectives of the proposed research are to determine the voltage dependence of C1- conductance in intact mature human red blood cells, and to characterize the properties of a C1- conducting channel observed when red cell vesicles are added to planar lipid bilayers under conditions which promote vesicle-bilayer fusion. Preliminary measurements of net K+ and C1- fluxes in intact red cells treated with valinomycin, in conjunction with determinations of membrane potential by fluorescent dyes, have suggested that C1- conductance is voltage-dependent, and further validation of this new observation is proposed. Along with additional controls, the distribution of radioactively labeled lipophilic ions will be used as an independent measure of membrane potentials. In addition, the characteristics of C1- selective, DIDS- sensitive, voltage-dependent channels incorporated into planar lipid bilayers upon apparent fusion of vesicles from human red cells will be ascertained. Subsequently, as another application of fluorescent membrane potential probes, their use in conjunction with flow cytometry for separating red cells according to membrane potential will be evaluated with mature red cells of known membrane potential, with cultured cells capable of induction to mature along the erythrocytic and granulocytic series, and with mixed marrow suspensions. These studies are intended to increase understanding of the mechanism of C1- transport across normal red cell membranes and also to develop new methodologies for comparing and understanding the differences between mature, immature, and abnormal human red blood cells. The proposed studies of the voltage dependence of C1- conductance is the main focus of the project and has the following significance: (1) the new information will constrain models for the mechanism of C1- transport; (2) the new data will have an important relationship to the question of whether C1- conductance in red cells is via """"""""slippage"""""""" through the anion exchanger or through an """"""""independent"""""""" pathway; (3) if the indications of voltage-dependent C1- conductance in red cells prove to be correct, the studies may help to increase understanding of voltage-dependent permeabilities in general; and (4) once the proposed method of measuring C1- conductance is established for normal mature red cells, then it will be possible in the future to reexamine indications of altered C1- conductance in red cells from patients with hemolytic anemias.
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