Substrate exchange properties of the high‐affinity glutamate transporter EAAT2

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.