Abstract
A stable cell line expressing the predominant brain glutamate transporter EAAT2 was used for the characterization of substrate exchange as a biochemical index for discriminating between substrate and non‐substrate inhibitors of the cloned EAAT2 transporter. Addition of 1 mM unlabeled D‐aspartate to cells equilibrated with [^3^H]D‐aspartate produced a time‐dependent depletion of the [^3^H] label retained by the cells. L‐Aspartate, L‐glutamate and L‐cysteate produced an equivalent degree of [^3^H] exchange to that observed with D‐aspartate, although the non‐substrate EAAT2 inhibitor dihydrokainate and D‐glutamate, which does not interact with the substrate binding site, failed to stimulate [^3^H]D‐aspartate exchange. Estimation of EC~50~ values for the stimulation of [^3^H] exchange by D‐aspartate, L‐glutamate and L‐trans‐2,4‐pyrollidine carboxylate (trans‐PDC) produced values that were in excellent agreement with the corresponding IC~50~ values for the same compounds to inhibit EAAT2 uptake. Moreover, trans‐PDC was found to produce a lower maximal exchange than that observed with D‐aspartate, consistent with the known partial EAAT2 substrate activity of trans‐PDC. The estimate of drug induced [^3^H] efflux with the cloned EAAT2 transporter represents a convenient biochemical assay for the discrimination of substrate and non‐substrate inhibitors of the EAAT2 subtype. J. Neurosci. Res. 66:482–486, 2001. © 2001 Wiley‐Liss, Inc.