Differentiation of substrate and nonsubstrate inhibitors of the high-affinity, sodium-dependent glutamate transporters

Within the mammalian central nervous system, the efficient removal of L-glu tamate from the extracellular space by excitatory amino acid transporters ( EAATs) has been postulated to contribute to signal termination, the recycli ng of transmitter, and the maintenance of L-glutamate at concentrations bel ow those that are excitotoxic. The development of potent and selective inhi bitors of the EAATs has contributed greatly to the understanding of the fun ctional roles of these transporters. In the present study, we use a library of conformationally constrained glutamate analogs to address two key issue s: the differentiation of substrates from nontransportable inhibitors and t he comparison of the pharmacological profile of synaptosomal uptake with th ose of the individual EAAT clones. We demonstrate that the process of trans porter-mediated heteroexchange can be exploited in synaptosomes to rapidly distinguish transportable from nontransportable inhibitors. Using this appr oach, we demonstrate that 2,4-methanopyrrolidine-2,4-dicarboxylate, cis-1-a minocyclobutane-1,3-dicarboxylate, and L-trans-2,4-pyrrolidine dicarboxylat e act as substrates for the rat forebrain synaptosomal glutamate uptake sys tem. In contrast, L-anti-endo-3,4-methanopyrrolidine-3,4-dicarboxylate, L-t rans-2,3-pyrrolidine dicarboxylate, and dihydrokainate proved to be competi tive inhibitors of D-[H-3] aspartate uptake that exhibited little or no act ivity as substrates. When these same compounds were characterized for subst rate activity by recording currents in voltage-clamped Xenopus laevis oocyt es expressing the human transporter clones EAAT1, EAAT2, or EAAT3, it was f ound that the pharmacological profile of the synaptosomal system exhibited the greatest similarity with the EAAT2 subtype, a transporter believed to b e expressed primarily on glial cells.