Effects of N-ethylmaleimide on ouabain-insensitive cation fluxes in human red cell ghosts

Abstract

In red cells of several species, the sulfhydryl reagent N-ethylmaleimide activates a Cl −-dependent, ouabain-resistant K + transport pathway. Here we report our attempts to demonstrate ouabain-resistant Cl −-dependent K + fluxes stimulated by N-ethylmaleimide in resealed human red cell ghosts using Rb + as a K + analogue. In contrast to intact cells, the rate constants of the base level Rb + efflux in ghosts were similar in NaNO 3 and NaCl (° k Rb = 0.535 ± 0.079 h −1 and 0.534 ± 0.085 h −1, respectively), while 1 mM N-ethylmaleimide stimulated Rb + efflux strongly in NaNO 3 (° k Rb = 14.26 ± 1.32 h −1) and moderately in NaCl (° k Rb = 2.73 ± 0.54 h −1). This effect was dependent on the presence of internal ATP. Stimulation of Rb + efflux was observed in the presence of ⩾ 0.2 mM N-ethylmaleimide and increased at pH values approaching 8.0, consistent with titration of SH groups. N-Ethylmaleimide-stimulated Rb + efflux was approx. 50% inhibited by 100 μM quinine sulfate whereas 1 μM bumetanide had no effect. In NaCl the N-ethylmaleimide-stimulated efflux saturated with initial internal ghost Rb + concentration, but rates increased linearly in NaNO 3. Replacement of external Na + with glucamine or choline decreased the N-ethylmaleimide-stimulated Rb + efflux, suggesting a role for external Na +. N-Ethylmaleimide-stimulated Rb + efflux was greater in buffers with lipophilic anions such as SCN − or NO 3 − than in solutions with Cl − or acetate. However, the conclude that the effect of N-ethylmaleimide on ouabain-resistant cation effluxes of human red cell ghosts is very different from the selective action of N-ethylmaleimide on Rb + influxes in intact red cells.