Contrasting Modes of Action of Methylglutamate Derivatives on the Excitatory Amino Acid Transporters, EAAT1 and EAAT2

Vandenberg, Robert J.; Mitrovic, Ann D.; Chebib, Mary; Balcar, Vladimír J.; Johnston, Graham A.R.

doi:10.1124/mol.51.5.809

Cited by 98 publications

(105 citation statements)

References 27 publications

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“…Hydropathy analysis revealed that these transporters would possess six membrane spanning helices in the NH 2 -terminal part and long hydrophobic stretches in the COOH-terminal half of the molecules (data not shown) as has been demonstrated for EAATs cloned to date [1,5,10,15,19]. These observations support recent findings that the COOH-terminal large hydrophobic regions of these subtypes, in common, consist of structural elements [7] that appear to be critical for coupling of the fluxes of Na + , K + and glutamate [11,23,26], although the precise membrane topology of this region remains controversial.…”

Section: Discussionsupporting

confidence: 79%

Cloning and Characterization of Excitatory Amino Acid Transporters GLT-1 and EAAC1 in Canine Brain.

Sato

Inaba

Baba

et al. 2001

The Journal of Veterinary Medical Science

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ABSTRACT. Excitatory amino acid transporters play important roles in termination of glutamatergic neurotransmission and protection of neurons from the excitotoxicity of glutamate in the central nervous system. We herein report isolation of cDNA clones of two distinct excitatory amino acid transporters, GLT-1 and EAAC1, from canine brain cortex by PCR-based cloning, and characterization of these transporter subtypes. Canine GLT-1 and EAAC1 exhibited Na + -dependent glutamate transport with high affinities in a Xenopus oocyte expression system. Despite the similarity in transport kinetics and in the predicted primary structures, GLT-1, EAAC1, and the previously identified GLAST showed different sensitivities to several structural analogues of L-glutamate. In addition, transcripts of these transporter subtypes showed distinct regional distribution in the brain in RT-PCR analysis, suggesting that excitatory amino acid transporters have distinct physiological and pathophysiological roles in the brain. KEY WORDS: brain, canine, cDNA cloning, excitatory amino acid transporter, glutamate.

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Section: Discussionsupporting

confidence: 79%

Cloning and Characterization of Excitatory Amino Acid Transporters GLT-1 and EAAC1 in Canine Brain.

Sato

Inaba

Baba

et al. 2001

The Journal of Veterinary Medical Science

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“…SYM-induced excitotoxicity in CGC was completely blocked by an NMDA receptor antagonist, MK-801, reminiscent with the finding that glutamate or domoic acid-induced Ca 2 þ influx and toxicity in CGC is preferentially blocked by NMDA receptor blockers. 21,22 Our results suggest that SYM-induced apoptosis is due to the accumulation of glutamate resulting from inhibition of EAATs 14 or enhanced glutamate release due to the activation of kainate receptors, 15,22 thus in turn Figure 5 VPA delays the accumulation of GAPDH in the nuclei of CGC following SYM treatment. (A) Immunohistochemistry of GAPDH.…”

Section: Discussionmentioning

confidence: 71%

“…However, the lack of kainite-induced death of CGC speaks against the latter possibility. 23 The role of EAATs in SYM-induced apoptosis is further supported by the observations that EAAT inhibitors exacerbate endogenous glutamate-induced excitotoxicity through NMDA receptor activation, 24 and that SYM is one of the most potent inhibitors of EAATs, 14 which are expressed in CGC neurons. 25,26 Our pioneering studies show that GAPDH overexpression and nuclear translocation is involved in apoptosis of several cell types subjected to various insults.…”

Section: Discussionmentioning

confidence: 92%

“…12 In addition, VPA induces axonal remodeling and synapsin clustering, presumably through indirect inhibition of glycogen synthase kinase-3 (GSK-3). 13 The present study was undertaken to investigate the neuroprotective effects of VPA and its underlying mechanisms against excitotoxicity induced by SYM 2081 ((2S, 4R)-4-methylglutamate), an inhibitor of excitatory amino-acid transporters (EAATs) 14 and an agonist of low-affinity kainate receptors, 15 in mature cerebellar granule cells (CGC). We also examined whether the neuroprotective effects of VPA were mimicked by inhibitors of HDAC and were associated with hyperacetylation of histone.…”

Section: Introductionmentioning

confidence: 99%

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Valproic acid inhibits histone deacetylase activity and suppresses excitotoxicity-induced GAPDH nuclear accumulation and apoptotic death in neurons

et al. 2004

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Valproic acid (VPA), used to treat bipolar mood disorder and seizures, also inhibits histone deacetylase (HDAC). Here, we found that VPA and other HDAC inhibitors, butyrate and trichostatin A, robustly protected mature cerebellar granule cell cultures from excitotoxicity induced by SYM 2081 ((2S, 4R)-4-methylglutamate), an inhibitor of excitatory amino-acid transporters and an agonist of low-affinity kainate receptors. These neuroprotective effects required protracted treatment and were correlated with enhanced acetylated histone levels, indicating HDAC inhibition. SYM-induced excitotoxicity was blocked by MK-801 ((5R,10S)-( þ )-5-methyl-10,11-dihydro-5H-dibenzo[a,d]-cyclohepten-5,10-imine hydrogen maleate), supporting that the toxicity was largely N-methyl-D-aspartate receptor dependent. SYM excitotoxicity had apoptotic characteristics and was prevented by a caspase inhibitor. SYMinduced apoptosis was associated with a rapid and robust nuclear accumulation of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a housekeeping gene previously shown to be proapoptotic. VPA pretreatment suppressed SYM 2081-induced GAPDH nuclear accumulation, concurrent with its neuroprotective effects. Chromatin immunoprecipitation (ChIP) revealed that GAPDH is copresent with acetylated histone H3, including Lys9-acetylated histone, and that VPA treatment caused a time-dependent decrease in the levels of nuclear GAPDH with a concomitant increase in acetylated histones in the ChIP complex. Our results strongly suggest that VPA protects neurons from excitotoxicity through inhibition of HDAC activity and that this protective effect may involve suppression of excitotoxicity-induced accumulation of GAPDH protein in the nucleus.

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“…Oocytes were harvested from X. laevis as described previously (16), and 50 nl of cRNA was injected into defoliculated stage V oocytes and incubated in standard frog Ringer's solution called ND96 (96 mM NaCl, 2 mM KCl, 1 mM MgCl 2 , 1.8 mM CaCl 2 , 5 mM HEPES, pH 7.55) supplemented with 2.5 mM sodium pyruvate, 0.5 mM theophylline, 50 g/ml gentamicin. Current recordings were made 2-5 days later using the two-electrode voltage clamp technique with a Geneclamp 500 amplifier (Axon instruments, Foster City, CA) interfaced with a MacLab 2e chart recorder (ADI Instruments, Sydney, Australia) using the Chart software and a Digidata 1200 (Axon Instruments), which is controlled by an IBM-compatible computer installed with pClamp software, version 7.0 (Axon Instruments).…”

Section: Methodsmentioning

confidence: 99%

Distinct Conformational States Mediate the Transport and Anion Channel Properties of the Glutamate Transporter EAAT-1

Ryan¹,

Vandenberg

2002

Journal of Biological Chemistry

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Glutamate transport by the excitatory amino acid transporters (EAATs) is coupled to the co-transport of 3 Na ؉ , 1 H ؉ , and the counter-transport of 1 K ؉ ion. In addition to coupled ion fluxes, glutamate and Na ؉ binding to the transporter activates a thermodynamically uncoupled anion conductance through the transporter. In this study, we have distinguished between these two conductance states of the EAAT-1 transporter using a [2-(trimethylammonium)ethyl]methanethiosulfonatemodified V452C mutant transporter. Glutamate binds to the modified mutant transporter and activates the uncoupled anion conductance but is not transported. The selective alteration of the transport function without altering the anion channel function of the V452C mutant transporter suggests that the two functions are generated by distinct conformational states of the transporter.Glutamate is the predominant excitatory neurotransmitter in the mammalian central nervous system, and the role of the excitatory amino acid transporters (EAATs) 1 is to regulate synaptic glutamate concentrations to maintain dynamic signaling between the presynaptic and postsynaptic membranes and also to prevent a build-up of toxic levels of glutamate. The energy used to drive the uphill transport of glutamate into the cell against a significant concentration gradient is derived from the coupling of transport to the co-transport of 3 Na ϩ and 1 H ϩ and the counter-transport of 1 K ϩ for each glutamate molecule (1, 2). In addition to these coupled ion fluxes, glutamate bound to the transporter activates a thermodynamically uncoupled anion conductance through the transporter (3-5).Five different glutamate transporters have been identified, and their amino acid sequences were determined from the cDNA sequences. The human glutamate transporters are termed EAAT-1 to 6,7), whereas the rat homologs of EAAT-1 and EAAT-2 are termed glutamate/aspartate transporter-1 (8) and glutamate transporter-1 (9) and the rabbit homolog of EAAT-3 is termed excitatory amino-acid carrier-1 (10). The five different transporter subtypes show a high degree of similarity and are likely to form similar structures in the membrane. A number of models for the membrane topology of glutamate transporters have been proposed. Initial predictions of the topology from hydrophobicity analysis of the amino acid sequence lead to three different models with 8, 10, and 12 transmembrane domains (8 -10), but more direct structural information has been obtained from method studies of substituted cysteine accessibility (11, 12). The models proposed from these studies both predict six ␣-helical transmembrane domains in the amino-terminal portion of the protein followed by a series of reentrant loops and membrane-associated structures followed by a final ␣-helical transmembrane domain and an intracellular carboxyl-terminal domain.The reentrant loop and membrane-associated structures are highly conserved in their amino acid sequences, and from mutagenesis studies, it has been proposed that this region forms the por...

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Contrasting Modes of Action of Methylglutamate Derivatives on the Excitatory Amino Acid Transporters, EAAT1 and EAAT2

Cited by 98 publications

References 27 publications

Cloning and Characterization of Excitatory Amino Acid Transporters GLT-1 and EAAC1 in Canine Brain.

Cloning and Characterization of Excitatory Amino Acid Transporters GLT-1 and EAAC1 in Canine Brain.

Valproic acid inhibits histone deacetylase activity and suppresses excitotoxicity-induced GAPDH nuclear accumulation and apoptotic death in neurons

Distinct Conformational States Mediate the Transport and Anion Channel Properties of the Glutamate Transporter EAAT-1

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