Substrate exchange properties of the high-affinity glutamate transporter EAAT2

A stable cell line expressing the predominant brain glutamate transporter E AAT2 was used for the characterization of substrate exchange as a biochemic al 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 [H-3]D-aspartate produced a time-dependent depletio n of the [H-3] label retained by the cells. L-Aspartate, L-glutamate and L- cysteate produced an equivalent degree of [H-3] exchange to that observed w ith D-aspartate, although the non-substrate EAAT2 inhibitor dihydrokainate and D-glutamate, which does not interact with the substrate binding site, f ailed to stimulate [H-3]D-aspartate exchange. Estimation of EC50 values for the stimulation of [H-3] exchange by D-aspartate, L-glutamate and L-trans- 2,4-pyrollidine carboxylate (trans-PDC) produced values that were in excell ent agreement with the corresponding IC50 values for the same compounds to inhibit EAAT2 uptake. Moreover, trans-PDC was found to produce a lower maxi mal exchange than that observed with D-aspartate, consistent with the known partial EAAT2 substrate activity of trans-PDC. The estimate of drug induce d [H-3] efflux with the cloned EAAT2 transporter represents a convenient bi ochemical assay for the discrimination of substrate and non-substrate inhib itors of the EAAT2 subtype. (C) 2001 Wiley-Liss, Inc.