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Glutamate transporters remove glutamate from the synaptic cleft to maintain efficient synaptic communication between neurons and to prevent glutamate concentrations from reaching neurotoxic levels. Glutamate transporters play an important role in ischemic neuronal death during stroke and have been implicated in epilepsy and amytropic lateral sclerosis. However, the molecular structure and the glutamate-uptake mechanism of these transporters are not well understood. The most recent models of glutamate transporters have three or five subunits, each with eight transmembrane domains, and one or two membrane-inserted loops. Here, using fluorescence resonance energy transfer (FRET) analysis, we have determined the relative position of the extracellular regions of these domains. Our results are consistent with a trimeric glutamate transporter with a large (>45 A) extracellular vestibule. In contrast to other transport proteins, our FRET measurements indicate that there are no large-scale motions in glutamate transporters and that glutamate uptake is accompanied by relatively small motions around the glutamate-binding sites. The large extracellular vestibule and the small-scale conformational changes could contribute to the fast kinetics predicted for glutamate transporters. Furthermore, we show that, despite the multimeric nature of glutamate transporters, the subunits function independently.

Glutamate transporters remove glutamate from the synaptic cleft to maintain efficient synaptic communication between neurons and to prevent extracellular glutamate concentrations from reaching neurotoxic levels (1). It is thought that glutamate transporters mediate glutamate transport through a reaction cycle with conformational changes between the two major access states that alternatively expose glutamate-binding sites to the extracellular or to the intracellular solution. However, there is no direct real-time evidence for the conformational changes predicted to occur during the transport cycle. In the present study, we used voltage-clamp fluorometry to measure conformational changes in the neuronal excitatory amino acid transporter (EAAT) 3 glutamate transporter covalently labeled with a fluorescent reporter group. Alterations in glutamate and cotransported ion concentrations or in the membrane voltage induced changes in the fluorescence that allowed detection of conformational rearrangements occurring during forward and reverse transport. In addition to the transition between the two major access states, our results show that there are significant Na(+)-dependent conformational changes preceding glutamate binding. We furthermore show that Na(+) and H(+) are cotransported with glutamate in the forward part of the transport cycle. The data further suggest that an increase in proton concentrations slows the reverse transport of glutamate, which may play a neuro-protective role during ischemia.

Within the mammalian central nervous system, the efficient removal of L-glutamate from the extracellular space by excitatory amino acid transporters (EAATs) has been postulated to contribute to signal termination, the recycling of transmitter, and the maintenance of L-glutamate at concentrations below those that are excitotoxic. The development of potent and selective inhibitors of the EAATs has contributed greatly to the understanding of the functional roles of these transporters. In the present study, we use a library of conformationally constrained glutamate analogs to address two key issues: the differentiation of substrates from nontransportable inhibitors and the comparison of the pharmacological profile of synaptosomal uptake with those of the individual EAAT clones. We demonstrate that the process of transporter-mediated heteroexchange can be exploited in synaptosomes to rapidly distinguish transportable from nontransportable inhibitors. Using this approach, we demonstrate that 2,4-methanopyrrolidine-2,4-dicarboxylate, cis-1-aminocyclobutane-1,3-dicarboxylate, and L-trans-2, 4-pyrrolidine dicarboxylate act as substrates for the rat forebrain synaptosomal glutamate uptake system. In contrast, L-anti-endo-3, 4-methanopyrrolidine-3,4-dicarboxylate, L-trans-2,3-pyrrolidine dicarboxylate, and dihydrokainate proved to be competitive inhibitors of D-[(3)H]aspartate uptake that exhibited little or no activity as substrates. When these same compounds were characterized for substrate activity by recording currents in voltage-clamped Xenopus laevis oocytes expressing the human transporter clones EAAT1, EAAT2, or EAAT3, it was found that the pharmacological profile of the synaptosomal system exhibited the greatest similarity with the EAAT2 subtype, a transporter believed to be expressed primarily on glial cells.