Characterization of Na(+)-dependent L-glutamate transport in canine erythrocytes

Characteristics of the high-affinity Na(+)-dependent L-glutamate transport system in canine erythrocytes were studied by using intact cells and resealed ghosts. The L-glutamate transport showed a precise dependence on extracellular Na+ and intracellular K+. Kinetical analysis revealed that two Na+ ions and one K+ ion were involved in each L-glutamate transport cycle. The L-glutamate transport was inhibited most potently by threo-3-hydroxyaspartate and L-cysteinesulfinate (at 25 microM, 83% and 79% inhibition, respectively) and weakly by dihydrokainate and DL-alpha-aminoadipate (at 25 microM, 21% and 17% inhibition, respectively). From these stoichiometrical and pharmacological properties we concluded that the L-glutamate transport system in canine erythrocytes is a product of the L-glutamate transporter gene family and resembles a neuronal transporter rather than a glial one. L-Glutamate uptake was increased by internal, but not external, HCO3- when the internal and external anions of the erythrocytes were replaced by several other anions. Moreover, this enhancement was blocked by inhibition of carbonic anhydrase, which indicated that L-glutamate transport was at least partly dependent on HCO3- generated inside the cells. These observations indicate that anion countertransport is coupled to the high-affinity Na(+)- and K(+)-dependent L-glutamate transport in canine erythrocytes.