title: Differentiation of substrate and nonsubstrate inhibitors of the high-affinity, sodium-dependent glutamate transporters.
abstract: 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.
authors.affiliation: Department of Pharmaceutical Sciences, University of Montana, Missoula, Montana, USA.
authors.forename: H P
authors.initials: HP
authors.lastname: Koch
authors.forename: M P
authors.initials: MP
authors.lastname: Kavanaugh
authors.forename: C S
authors.initials: CS
authors.lastname: Esslinger
authors.forename: N
authors.initials: N
authors.lastname: Zerangue
authors.forename: J M
authors.initials: JM
authors.lastname: Humphrey
authors.forename: S G
authors.initials: SG
authors.lastname: Amara
authors.forename: A R
authors.initials: AR
authors.lastname: Chamberlin
authors.forename: R J
authors.initials: RJ
authors.lastname: Bridges
mesh_terms: D027322:Amino Acid Transport System X-AG; D000818:Animals; D001224:Aspartic Acid / Q000378:metabolism; D001667:Binding, Competitive; D001692:Biological Transport; D002352:Carrier Proteins / Q000037:antagonists & inhibitors*; D027341:Excitatory Amino Acid Transporter 1; D027342:Excitatory Amino Acid Transporter 2; D050580:Excitatory Amino Acid Transporter 3; D050491:Glutamate Plasma Membrane Transport Proteins; D018698:Glutamic Acid / Q000031:analogs & derivatives / Q000494:pharmacology*; D066298:In Vitro Techniques; D008958:Models, Molecular; D009457:Neuroglia / Q000378:metabolism; D009865:Oocytes / Q000378:metabolism; D016548:Prosencephalon / Q000378:metabolism; D020033:Protein Isoforms / Q000378:metabolism; D051381:Rats; D017981:Receptors, Neurotransmitter / Q000737:chemistry / Q000378:metabolism; D027981:Symporters; D013574:Synaptosomes / Q000187:drug effects* / Q000378:metabolism; D014316:Tritium; D014982:Xenopus laevis
chemical_list: D027322:Amino Acid Transport System X-AG; D002352:Carrier Proteins; D027341:Excitatory Amino Acid Transporter 1; D027342:Excitatory Amino Acid Transporter 2; D050580:Excitatory Amino Acid Transporter 3; D050491:Glutamate Plasma Membrane Transport Proteins; D020033:Protein Isoforms; D017981:Receptors, Neurotransmitter; C493398:Slc1a1 protein, rat; C493395:Slc1a2 protein, rat; C493392:Slc1a3 protein, rat; D027981:Symporters; D014316:Tritium; D001224:Aspartic Acid; D018698:Glutamic Acid