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 [3H]D-aspartate produced a time-dependent depletion of the [3H] label retained by the cells. L-Aspartate, L-glutamate and L-cysteate produced an equivalent degree of [3H] 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 [3H]D-aspartate exchange. Estimation of EC50 values for the stimulation of [3H] 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 IC50 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 [3H] efflux with the cloned EAAT2 transporter represents a convenient biochemical assay for the discrimination of substrate and non-substrate inhibitors of the EAAT2 subtype.
Copyright 2001 Wiley-Liss, Inc.