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Actions of a carbocyanine dye on calcium-dependent potassium transport in human red cell ghosts
Abstract:
1. 3,3'-Diethylthiadicarbocyanine iodide (diS-C2-(5)), a fluorescent dye widely used to estimate membrane potentials, inhibits the Ca-dependent K transport system of human red cells and ghosts. It does not affect non-specific cation leaks, net chloride movements or the Ca-activated ATPase. 2. The nature of the inhibitory effect depends upon the conditions under which the dye is applied. When Ca-containing ghosts with 100 mM internal K are suspended in a choline medium at 37 degrees C, 0.3 microM-diS-C2-(5) causes virtually complete inhibition of K transport within 1 min (haematocrit 0.3%). Inhibition cannot be reversed by washing the ghosts to remove bound dye, although partial recovery occurs on subsequent incubation at 37 degrees C. 3. Inhibition is less marked when ghosts are suspended in solutions containing 100 mM-K, and is largely reversed by washing. The degree of inhibition varies with the dye concentration: 50% inhibition occurs with a free diS-C2-(5) concentration of 0.7 microM, and about 5 x 10(6) dye molecules are bound per ghost. 4. Changes in internal and external K concentrations, keeping internal Ca constant, have no effect on the dye concentration required for 50% inhibition of K efflux. 5. When internal Ca is varied, keeping internal and external K constant, the dye concentration needed for 50% inhibition of K efflux varies inversely with the K efflux in the absence of dye. That is, diS-C2-(5) is most effective as an inhibitor when K efflux is at a maximum. This suggests that the dye reacts preferentially with the transport system in the Ca-activated state. 6. The effects of several related molecules were examined, using Rb influx as a measure of K permeability. Inhibition is obtained in some cases, but is not directly related to the ability of the ghosts to bind the dyes. An anionic dye, diS-C3SO3--(5) inhibits Rb influx only when present inside the ghosts. 7. The implications of these findings are discussed.