Differentiation of Substrate and Nonsubstrate Inhibitors of the High-Affinity, Sodium-Dependent Glutamate Transporters1
- Hans P. Koch1,
- Michael P. Kavanaugh3,
- Christopher S. Esslinger2,
- Noah Zerangue3,
- John M. Humphrey4,
- Susan G. Amara3,5,
- A. Richard Chamberlin4 and
- Richard J. Bridges1
- Departments of 1Pharmaceutical Sciences (H.P.K., R.J.B.) and2Chemistry (C.S.E.), University of Montana, Missoula, Montana;3Vollum Institute, Oregon Health Sciences University, Portland, Oregon (M.P.K., N.Z., S.G.A.); 4Department of Chemistry, University of California, Irvine, California (J.M.H., A.R.C.); and 5Howard Hughes Medical Institute, Portland, Oregon (S.G.A.)
Abstract
Within the mammalian central nervous system, the efficient removal ofl-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, andl-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 ofd-[3H]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.
Footnotes
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Send reprint requests to: Dr. Richard J. Bridges, Department of Pharmaceutical Sciences, School of Pharmacy and Allied Health Sciences, University of Montana, Missoula, Montana. E-mail:bridgesr{at}selway.umt.edu
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↵1 This work has been presented in part in abstract form inSoc Neurosci Abstr (1995) 22:1574.
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This work was supported in part by National Institutes of Health Grants NS30570 (R.J.B.), NS33270 (M.P.K.), NS27600 (A.R.C.), NS10156 (C.S.E.), and NS33272 (S.G.A.).
- Abbreviations:
- CNS
- central nervous system
- EAA
- excitatory amino acid
- EAAT
- excitatory amino acid transporter
- PDC
- pyrrolidine dicarboxylate
- KA
- kainate
- DHK
- dihydrokainate
- ACBD
- 1-aminocyclobutane-1,3-dicarboxylate
- MPDC
- methano-pyrrolidine-2,4-dicarboxylate
- THA
- threo-hydroxyaspartate
- ADC
- azetidine-2,3-dicarboxylate
- cis-5-Me-l-trans-2,3-PDC
- (2S,3S,5S)-5-methyl-pyrrolidine dicarboxylate
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- Received May 3, 1999.
- Accepted August 12, 1999.
- The American Society for Pharmacology and Experimental Therapeutics
