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Vol. 53, Issue 2, 195-201, February 1998
-Benzyloxyaspartate, A
Potent Blocker of Excitatory Amino Acid Transporters
Suntory Institute for Bioorganic Research, Wakayamadai, Shimamoto-cho, Mishima-gun, Osaka 618, Japan (K.S., B.L., Y.Y-K., M.S., T.N), and Osaka National Research Institute (Agency of Industrial Science and Technology, Ministry of International Trade and Industry), Midorigaoka, Ikeda, Osaka 563, Japan (Y.S., N.Y.)
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Summary |
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Summary
Introduction
Procedures
Results
Discussion
References
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DL-threo-
-Benzyloxyaspartate
(DL-TBOA), a novel derivative of
DL-threo--hydroxyaspartate, was
synthesized and examined as an inhibitor of sodium-dependent
glutamate/aspartate (excitatory amino acid) transporters.
DL-TBOA inhibited the uptake of
[14C]glutamate in COS-1 cells expressing the human
excitatory amino acid transporter-1 (EAAT1)
(Ki = 42 µM)
with almost the same potency as
DL-threo--hydroxyaspartate
(Ki = 58 µM).
With regard to the human excitatory amino acid transporter-2 (EAAT2),
the inhibitory effect of DL-TBOA
(Ki = 5.7 µM) was much more potent than that of dihydrokainate
(Ki = 79 µM),
which is well known as a selective blocker of this subtype. Electrophysiologically, DL-TBOA induced no detectable
inward currents in Xenopus laevis oocytes expressing
human EAAT1 or EAAT2. However, it significantly reduced the
glutamate-induced currents, indicating the prevention of transport. The
dose-response curve of glutamate was shifted by adding
DL-TBOA without a significant change in the maximum
current. The Kb values for human
EAAT1 and EAAT2 expressed in X. laevis oocytes were 9.0 µM and 116 nM, respectively. These
results demonstrated that DL-TBOA is, so far, the most
potent competitive blocker of glutamate transporters.
DL-TBOA did not show any significant effects on either
the ionotropic or metabotropic glutamate receptors. Moreover,
DL-TBOA is chemically much more stable than its
benzoyl analog, a previously reported blocker of excitatory amino acid
transporters; therefore, DL-TBOA should be a useful
tool for investigating the physiological roles of transporters.
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Introduction |
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Summary
Introduction
Procedures
Results
Discussion
References
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Glutamate
acts as an excitatory neurotransmitter in the mammalian central nervous
system as well as a potent neurotoxin. The termination of
neurotransmission is mediated by sodium-dependent high affinity
glutamate/aspartate transporters. Glutamate transporters also play an
important role in maintaining the extracellular glutamate concentration
below neurotoxic levels and therefore contribute to the prevention of
neuronal damage from excessive activation of glutamate receptors
(Nicholls and Attwell, 1990
; Rothstein et al., 1996;
Gegelashvili and Schousboe, 1997; Kanai, 1997; Tanaka et
al., 1997). Some serious neuronal diseases, such as epilepsy, amyotrophic lateral sclerosis, Alzheimer's diseases, and cellular damage from a stroke, may be linked to the failure of transporters. Five subtypes of EAATs (EAAT1-5) have been cloned from mammalian tissues (Kanai and Hediger, 1992; Pines et al., 1992; Storck
et al., 1992; Tanaka, 1993; Shashidharan et al.,
1993, 1994; Arriza et al., 1994, 1997; Kawakami et
al., 1994; Manfras et al., 1994; Fairman et
al., 1995; Inoue et al., 1995). These transporters couple the electrochemical gradient of three cotransported sodium ions
and one countertransported potassium ion to that of glutamate (Zerangue
and Kavanaugh, 1996). A proton also is cotransported. In addition, a
substrate-dependent chloride conductance provides a potential mechanism
for dampening cell excitability (Fairman et al., 1995). The
transporter subtypes notably differ in the magnitude of this chloride
flux relative to the flux of glutamate (Fairman et al.,
1995; Wadiche et al., 1995; Arriza et al., 1997).
Inhibitors of glutamate transporters are essential for elucidation of
the intrinsic properties and physiological roles of transporters. A
number of pharmacological agents have been shown to inhibit glutamate
transport (Ferkany and Coyle, 1986; Bridges et al., 1991,
1993, 1994; Nakamura et al., 1993; Robinson et
al., 1993; Arriza et al., 1994; Yamashita et
al., 1995; Vandenberg et al., 1997). Most of them
indeed act as competitive substrates, inducing a transport current and
a substrate-dependent chloride flux. Blocker-type inhibitors, which are
not transportable, inhibit the transport of glutamate while letting
transporters to be electrically silent. Moreover, they also suppress
voltage-dependent pre-steady state charge movements, allowing kinetic
information on the transporters to be obtained (Wadiche et
al., 1995). KA and DHKA block EAAT2 in the low-micromolar range,
whereas 
10 mM is required to block transport by EAAT1,
EAAT3, and EAAT4 (Arriza et al., 1994; Fairman et
al., 1995). Recently, 2S4R4MG was shown to be a very potent blocker for EAAT2 and a substrate for EAAT1 (Vandenberg et
al., 1997). However, these compounds also activate ionotropic
glutamate receptors (Gu et al., 1995). As such, they can be
valuable pharmacological tools for the study of EAAT2 in heterologous
expression systems but cannot be used to determine the physiological
role of this transporter in complex preparations. THA and
t-2,4-PDC were found to be blockers for EAAT5 (Arriza
et al., 1997) and substrates for EAAT1-4 (Arriza et
al., 1994; Fairman et al., 1995). THA also was
demonstrated to be a ligand for
N-methyl-D-aspartate receptors (Jane et
al., 1994); therefore, pharmacological agents that are able to
block EAATs without being transported and without affecting glutamate
receptors are needed.
Our approach to the development of blockers of EAATs was the synthesis
of derivatives of THA. We recently demonstrated that DL-TBzOAsp is a competitive blocker for bovine EAAT1
(glutamate/aspartate transporter type) (Lebrun et al.,
1997). However, because DL-TBzOAsp has an ester bond, it is
unstable in aqueous solution (ester cleavage or acyl migration);
therefore, we synthesized a more stable ether-type derivative,
DL-TBOA. DL-TBOA showed potent inhibitory
activity on the [14C]glutamate uptake in COS-1
cells expressing human EAAT1 or EAAT2. Moreover, it proved to be highly
selective for EAATs versus the glutamate receptors.
Electrophysiological analysis demonstrated that DL-TBOA is
a competitive blocker for human EAAT1 and EAAT2; it is indeed the most
potent blocker for both EAAT1 and EAAT2 described to date.
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Experimental Procedures |
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Summary
Introduction
Procedures
Results
Discussion
References
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Materials.
L-Glutamate was obtained from Nacalai
Tesque (Kyoto, Japan). THA was from Sigma Chemical (St. Louis, MO).
t-2,4-PDC, DHKA, and 2S4R4MG were from Tocris Cookson
(Bristol, UK). L-[14C]Glutamate,
[3H]CGS 19755 (cis-4-phosphono-methyl-2-piperidine carboxylic acid), [3H]KA, and [3H]AMPA
were from DuPont-New England Nuclear (Botson, MA).
L-CCG-III and L-CCG-IV were synthesized as
described previously (Shimamoto et al., 1991).
DL-TBOA was synthesized in the same manner as
DL-TBzOAsp, except for the use of benzyl bromide instead of
acyl chloride (Lebrun et al., 1997). The structure and
purity (>95%) of the compound were confirmed with 400-MHz NMR. Stock
solutions (100 mM) of the inhibitors, except for
DL-TBzOAsp, were made in 0.1 M NaOH and stored
at 
20°. Stock solutions for DL-TBzOAsp were made in
50% dimethylsulfoxide without NaOH. DL-TBOA was stable for
1 week at room temperature; no noticeable decomposition was observed
by NMR.
cDNA cloning.
The cDNA clones coding for the human EAAT1 or
EAAT2 were obtained by PCR performed on human brain cDNA (Clontech,
Palo Alto, CA) using Ex Taq polymerase (Takara Shuzo, Shiga,
Japan). To amplify the full-length EAAT-coding regions, sets of primers
were designed according to the published nucleotide sequences
(Shashidharan et al., 1993, 1994; Arriza et al.,
1994; Kawakami et al., 1994; Manfras et al.,
1994) (set for EAAT1,
5
-AGCTGGAGCTCCACCCCTTACAAAATCAGAAAAGTTGTGTTTTC-3 and
5-AATTGGGTACCTGGTGCTCAAGAAAGTGTTTCTTTATGTTAGTC-3; set for EAAT2,
5-AGCTGGAGCTCACCCCGGCGTCCGCTTTCTCCCTCGCCCACATC-3 and 5-AATTGGGTACCATAGGATACGCTGGGGAGTTTATTCAAGAATTTG-3). Thirty
cycles of amplification were carried out using a thermal cycle program (EAAT1, 1 min at 94°, 1 min at 55°, and 1 min at 72°; EAAT2, 1 min at 94°, 1 min at 45°, and 1 min at 72°). The PCR products (1.8 kbp for EAAT1 and 1.9 kbp for EAAT2) were cloned into pCR II
according to the manufacturer's instruction for the Original TA
Cloning Kit (InVitrogen, San Diego, CA). The obtained clones were
subsequently analyzed by DNA sequencing. The clones containing EAATs
were subcloned into pBluescript II SK
after
digestion with SacI and KpnI for expression in
Xenopus laevis oocytes. The plasmids were linearized with
BssHII, and cRNA was transcribed from each of the cDNA
constructs with T3 RNA polymerase and capped using the MEGAscript kits
and Cap Analog (Ambion, Austin, TX). The eukaryotic expression vector
pKDEMSS (Kitano et al., 1995) derived from pdKCR-dhfr was
kindly donated by Dr. K. Kitano (Suntory Institute for Biomedical
Research, Mishima-gun, Osaka, Japan). EAAT1 cloned in pCR II was
digested with SpeI and XhoI and subcloned into
the SpeI/SalI sites of pKDEMSS. EAAT2 cloned in
pCR II was subcloned into pKDEMSS after digestion with EcoRI.
Transfection. COS-1 cells were cultured in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum at 37° under an atmosphere of 5% CO2. Cells were transfected by electroporation (1 × 107 cells, 200 V, 975 µF) with 10 µg of the eukaryotic expression vector pKDEMSS containing each type of transporter cDNA. COS-1 cells transfected by the vector alone were used to control the level of the endogenous uptake of [14C]glutamate. Transfected cells were seeded onto 96-well plates and cultured for 2 days before the uptake assay.
Measurements of [14C]glutamate uptake in
transfected COS-1 cells.
The subconfluent cells were washed two
times with 300 µl of modified phosphate-buffered saline that
contained 137 mM NaCl, 2.7 mM KCl, 8.1 mM Na2HPO4, 1.5 mM KH2PO4, 1 mM MgCl2, 1 mM
CaCl2, and 10 mM
D-glucose, pH 7.4 (Yamashita et al., 1995) and
preincubated in 300 µl of the same buffer at 37° for 12 min. After
aspiration of the buffer, cells were incubated with 1 µM
L-[14C]glutamate in 100 µl of
modified phosphate-buffered saline in the absence or presence of test
compounds at various concentrations at 37° for 12 min. To terminate
the uptake, cells were washed three times with ice-cold buffer and
solubilized with 100 µl of 1 M NaOH. Radioactivity was
measured by scintillation counting in 3 ml of ULTIMAGOLD (Packard,
Meriden, CT). Nonspecific incorporation was determined in sodium-free
solution (140 mM choline chloride, 5 mM KCl, 1 mM CaCl2, 1 mM
MgCl2, 20 mM HEPES, and 10 mM D-glucose, pH 7.4) (Nakamura et
al., 1993). Specific uptake of
[14C]glutamate is given relative to the
control. The inhibition constants (Ki
values) were determined by Dixon and/or Lineweaver-Burk plots obtained
for increasing concentrations of [14C]glutamate
of 0.5-5 µM. All values displayed are
mean ± standard error of at least three determinations.
Expression of EAAT in oocytes. Fifty nanograms of RNA coding for EAAT1 or EAAT2 was injected into stage V-VI defolliculated oocytes.
Electrophysiology.
Two-electrode voltage-clamp recordings
were made 2-4 days after injection with the use of the same apparatus
and methods as described previously (Lebrun et al., 1997).
Receptor binding assay.
Rat brain synaptic membranes were
prepared (Enna and Snyder, 1977) and modified (Murphy et
al., 1988) as described previously and stored at 80° until
use. On the day of the assay, the membrane suspension was incubated in
a buffer containing 0.04% Triton X-100 at 37° for 30 min (Murphy
et al., 1987, 1988). Triton X-100 was removed by
centrifugation, and the pellet was washed twice with an assay buffer.
Binding assays were performed according to published methods (London
and Coyle, 1979; Murphy et al., 1987, 1988; Kawai et
al., 1992). Incubation conditions are described in the legend for
Table 3.
mGluR assay.
CHO cells stably expressing mGluR1, mGluR2, or
mGluR4 were kindly donated by Prof. S. Nakanishi (Kyoto University,
Kyoto, Japan). Agonist and antagonist activities on mGluR1 were
determined by intracellular Ca2+ concentration
measurements on Fura-2/acetoxymethyl ester-loaded cells in the absence
or presence of glutamate (10 µM), respectively, as
described previously (Kawabata et al., 1996). Agonist
activities on mGluR2 or mGluR4 were evaluated by measuring the
inhibition of forskolin-induced cAMP formation as described previously
(Hayashi et al., 1992). Antagonist activities were evaluated
by measuring the rescue from the glutamate (10 µM)-induced inhibition of forskolin-induced cAMP
formation.
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Results |
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Top
Summary
Introduction
Procedures
Results
Discussion
References
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We recently obtained the first competitive blockers for bovine
EAAT1 (glutamate/aspartate transporter) by introducing some bulky acyl
substituents on the -hydroxyl group of DL-THA (Lebrun et al., 1997). However, these derivatives are unstable in
aqueous solution, particularly at basic pH, due to the presence of an easily cleavable ester bond and a possible intramolecular acyl migration from oxygen to nitrogen. Furthermore, they might be susceptible to esterases in biological preparations. To obtain a more
stable derivative, we synthesized the ether-type analog DL-TBOA (Fig. 1). This new
compound proved to be stable even in a basic solution (disodium salt,
pH 11.5) at room temperature. Because DL-TBOA was
insensitive to the pH of a solution, it could be treated as a
water-soluble sodium salt.
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Cloning of human EAAT1 and EAAT2.
To compare the inhibitory
action of DL-TBOA with those of DL-TBzOAsp, our
most potent blocker for EAAT1, and other known inhibitors on human
EAAT1 and EAAT2, we first cloned these transporters by PCR from a human
brain cDNA library. The sequence of our EAAT1 cDNA clone was identical
to those of GenBank nos. D26443 (Kawakami et al., 1994), L19158
(Shashidharan and Plaitakis, 1993), and U03504 (Arriza et
al., 1994). On the contrary, there were some nucleotide
differences between our EAAT2 cDNA clone and those of GenBank nos.
U03505 (Arriza et al., 1994), U01824 (Shashidharan et
al., 1994), and Z32517 (Manfras et al., 1994). The
nucleotide sequence of our clone has been submitted to the
GenBank/EMBL/DDBJ Data Bank with accession no. D85884. The predicted
human EAAT2 gene product reported here has 99% sequence identity with
that of U03505, 98% with that of U01824, and 92% with that of Z32517.
Three amino acids (His19, Val211, and Phe347) in the amino acid
sequence predicted from our clone were replaced in a very similar
sequence predicted from UO3505, whereas these amino acids were
conserved in the sequences predicted by the other clones; therefore,
the differences do not seem to be errors introduced by the
Taq polymerase. Because the characteristics of EAAT2
described below were very similar to those reported previously, we
postulate that the substitutions have little effect on the transporter
activity.
Inhibition of radiolabeled glutamate uptake in transfected COS-1 cells. Transfections were performed according to the electroporation method as described in Experimental Procedures. Transfected cells accumulated 5-10-fold more [14C]glutamate than vector-transfected cells. Uptake was reduced by >90% in Na+-free solution. Michaelis constant (Km) values for L-glutamate were 57 ± 6.0 µM for EAAT1 and 49 ± 10 µM for EAAT2.
Both DL-TBOA and DL-TBzOAsp markedly inhibited [14C]glutamate (1 µM) uptake in transfected cells in a dose-dependent manner (Fig. 2). On EAAT1, DL-TBOA was more potent than DL-TBzOAsp, with almost the same level of activity as that of DL-THA. Values of 50% inhibitory concentration (IC50) were 67 µM for DL-TBOA, 286 µM for DL-TBzOAsp, and 96 µM for DL-THA. Some other compounds, well known inhibitors acting as competitive substrates, were examined in the same system (Table 1). Among them, L-CCG-III very significantly inhibited [14C]glutamate uptake with an IC50 value of 11 µM, which is in accord with previous studies (Kawai et al., 1992; Nakamura
et al., 1993; Yamashita et al., 1995).
L-CCG-IV, a stereoisomer of L-CCG-III, was
reported to be nearly as potent as L-CCG-III on human
glutamate transporter-1 expressed in COS-7 cells (Yamashita et
al., 1995). It showed only a weak affinity
(IC50 = 900 µM) in this work, which is consistent with its poor inhibitory activity on the uptake by the
glial plasmalemmal vesicle and synaptosomes (Kawai et al., 1992; Nakamura et al., 1993). On EAAT2, the inhibitory
effect of DL-TBOA was much stronger than that of
DL-TBzOAsp and DL-THA (IC50 = 5.5, 126, and 31 µM,
respectively). The potency of DL-TBOA was comparable to
that of L-CCG-III (IC50 = 4.6 µM). DHKA, known to be a selective blocker of EAAT2,
inhibited the uptake on EAAT2 with an IC50 value
of 196 µM, whereas it had no effect on EAAT1 at a
concentration as high as 1 mM. Recently, 2S4R4MG was
reported to be the most potent blocker for EAAT2 and a substrate for
EAAT1 in an electrophysiological study using X. laevis
oocytes expressing EAAT1 or EAAT2 (Vandenberg et al., 1997).
In this study, 2S4R4MG (IC50 = 69 µM) proved to be more potent than DHKA on EAAT2 but less
potent than DL-TBOA.
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Electrophysiological studies on X. laevis oocytes. To determine whether DL-TBOA is a substrate or a blocker of EAAT1 and EAAT2, we performed an electrophysiological analysis of its effect on X. laevis oocytes injected with cRNA encoding EAAT1 or EAAT2. DL-TBOA (100 µM) did not elicit a detectable current in voltage-clamped oocytes expressing EAAT1 or EAAT2. However, the inward current induced by glutamate (100 µM) was significantly reduced in the presence of DL-TBOA (100 µM) (Fig. 4).
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1) was plotted against log[DL-TBOA]
(Fig. 5C). Antagonism equilibrium dissociation constants
(Kb values) of 9.0 µM for EAAT1 and 116 nM
for EAAT2 were obtained from a linear fit of the data with slopes of
1.01 and 1.06, respectively.
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Activity on ionotropic glutamate receptors and mGluRs. A key requirement for blockers of EAATs to be valuable pharmacological tools is selectivity toward EAATs versus glutamate receptors. We analyzed the activity of our derivatives on the ionotropic glutamate receptors by binding assays and on the mGluRs by calcium influx measurements (mGluR1) or cAMP formation monitoring (mGluR2 and mGluR4). DL-TBOA showed only a weak affinity toward ionotropic glutamate receptors. In binding competition with [3H]CGS 19755 (N-methyl-D-aspartate-type antagonist), [3H]KA, and [3H]AMPA on rat brain synaptic membranes, the IC50 values of DL-TBOA were 472 ± 139 µM, 550 ± 250 µM, and > 1 mM, respectively (Table 3). DL-TBOA did not show any agonist or antagonist activity on mGluRs (mGluR1, mGluR2, and mGluR4) at a concentration of 100 µM (data not shown).
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Discussion |
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Top
Summary
Introduction
Procedures
Results
Discussion
References
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The results of this study demonstrated that DL-TBOA is a potent competitive blocker of EAAT1 and EAAT2. Furthermore, it showed no activity on the metabotropic receptors tested so far (mGluR1, mGluR2, and mGluR4) at a concentration of 100 µM and was only weakly active on the ionotropic receptors. Thus, this compound proved to be highly selective for EAATs versus the glutamate receptors. The previously described blockers of EAAT2 lacked such a selectivity. KA and 2S4R4MG are high affinity ligands for KA receptors, and DHKA is active on both KA receptors and EAAT2 within the same concentration range; therefore, DL-TBOA will be the blocker of choice for studying the physiological roles of EAATs.
We determined the kinetic parameters of DL-TBOA on EAAT1
and EAAT2 in two different assay systems: the uptake in COS-1 cells and
the voltage-clamp study in X. laevis oocytes. It should be noted that the Ki value for
DL-TBOA obtained from the uptake assay was
significantly higher than the Kb
value from the electrophysiological assay. The discrepancy was lower in
the case of EAAT1 (Ki = 42 µM, Kb = 9 µM) than in the case of EAAT2
(Ki = 5.7 µM,
Kb = 0.12 µM). The Kb
values for DHKA and 2S4R4MG on EAAT2 were determined previously (9 and
3.4 µM, respectively) with a
electrophysiological study using X. laevis oocytes (Arriza
et al., 1994; Vandenberg et al., 1997). Their
affinity in COS-1 cells were also tested and proved to be
underestimated. System differences might be attributed to differences
in lipid composition, post-translational modification, and other
factors (Arriza et al., 1994). Of importance could be that
COS-1 cells are not voltage-clamped as opposed to X. laevis oocytes. Presumably, transport over a 12-min interval would cause a
significant depolarization and, as such, would slow the transport rate.
The blocker could suppress such a depolarization and restore the
transport rate. The control cells that take up glutamate would be more
depolarized than the blocked ones; therefore, the relative measurement
would lead to underestimation of the apparent activity of blockers.
EAAT1 displays a stronger substrate-dependent chloride conductance than
EAAT2 (Wadiche et al., 1995). This chloride conductance may
reduce the membrane depolarization and therefore limit the underestimation of the affinity for EAAT1. We nevertheless acknowledge that Arriza et al. (1994) observed a lower discrepancy in
the affinity of DHKA on EAAT2 expressed in COS-7 cells
(Ki = 23 µM) versus in X. laevis oocytes
(Kb = 9 µM).
It would be plausible that a particular conformation of glutamate or
aspartate can be recognized by the substrate-binding sites of
transporters. TBOA is a flexible molecule that can adopt several
conformations. For the development of selective blockers, it is to
elucidate the active conformation for binding to EAATs. In recent
studies, assessment was attempted of the conformational requirements
for glutamate binding to EAATs (Shimamoto et al., 1991;
Bridges et al., 1991, 1993, 1994; Kawai et al.,
1992; Nakamura et al., 1993; Yamashita et al.,
1995; Vandenberg et al., 1997). Bridges et al.
(1991, 1993, 1994) proposed that a folded form of glutamate binds to
EAATs while an extended form of aspartate is adopted. This hypothesis
seemed to be reasonable because the functional groups (two carboxylates
and an amino group) of these conformers and those of the active
compounds can be well superimposed (Bridges et al., 1991,
1993, 1994). Thus, we assumed that the active conformation of TBOA
would be an extended one.
Besides the conformational requirements described above, the
diastereomeric properties of the compounds are recognized by transporters. L-Glutamate is a high affinity substrate,
whereas D-glutamate is poorly transported. On the other
hand, both enantiomers of aspartate are known to be excellent
substrates of EAATs. Moreover, four diastereomers of
-hydroxyaspartate (L- and D-, and
threo- and erythro-) were nearly equal in
activity (Robinson et al., 1993). We suggest that such a
permissiveness in structure could be lost in TBOA, in which the small
hydroxyl group of THA has been replaced by a bulky benzyl group. We
propose that the bulky substituent in TBOA plays a very important role
in conferring blocking activity, whereas the hydroxyl of THA is simply
tolerated in the pharmacophore (Lebrun et al., 1997).
Therefore, the orientation of this substituent relative to the
aspartate framework should be very important. The synthesis of the pure
stereoisomers of TBOA and erythro-isomers is under way. The
elucidation of the binding conformation and blockade mechanism might
make it possible to develop more potent, and perhaps more selective,
EAAT blockers.
So far, DL-TBOA has proved to be the most potent blocker for EAAT1 and EAAT2, with high selectivity toward EAATs versus the glutamate receptors. Moreover, our preliminary results in this electrophysiological study indicated that DL-TBOA also was a blocker for the mouse EAAC1 (EAAT3 subtype). Therefore, this compound opens the way for the mechanistic and kinetic studies of EAAT1 and EAAT3. It should prove useful in determination of the physiological roles of EAATs in various preparations.
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Acknowledgments |
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We wish to thank Prof. Y. Kanai (Kyorin University, Tokyo, Japan) for providing the cDNA of mouse EAAC1, and Prof. S. Nakanishi (Kyoto University, Kyoto, Japan) for supplying the CHO cells expressing mGluRs. We also thank Dr. M. Fujimoto and Dr. T. Nakano (Shionogi Co., Osaka, Japan) for assistance with the mGluR assay.
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Footnotes |
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Received October 6, 1997; Accepted November 4, 1997
Send reprint requests to: Dr. Keiko Shimamoto, Suntory Institute for Bioorganic Research, 1-1-1, Wakayamadai, Shimamoto-cho, Mishima-gun, Osaka 618, Japan. E-mail: ldf01705{at}niftyserve.or.jp
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Abbreviations |
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EAAT, excitatory amino acid transporter;
EAAC, excitatory amino acid carrier;
THA, threo--hydroxyaspartate;
TBzOAsp, threo--benzoyloxyaspartate;
TBOA, threo--benzyloxyaspartate;
t-2, 4-PDC,
L-trans-pyrrolidine-2,4-dicarboxylic acid;
L-CCG-III, (2S,1S,2R)-2-(2-carboxycyclopropyl)glycine;
L-CCG-IV, (2S,1R,2S)-2-(2-carboxycyclopropyl)glycine;
2S4R4MG, (2S,4R)-4-methylglutamate;
PCR, polymerase chain reaction;
DHKA, dihydrokainate;
KA, kainate;
AMPA, 
-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid;
mGluR, metabotropic glutamate receptor.
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References |
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Top
Summary
Introduction
Procedures
Results
Discussion
References
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-aminobutyric acid receptor binding in synaptic membranes of rat brain.
Mol Pharmacol
13:
442-453-hydroxyaspartate derivatives are competitive antagonists for bovine glutamate/aspartate transporter.
J Biol Chem
272:
20336-20339-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) and a filtration assay.
Neurochem Res
12:
775-782[Medline]. This article has been cited by other articles:
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M.-H. Kim, S. Uehara, A. Muroyama, B. Hille, Y. Moriyama, and D.-S. Koh Glutamate Transporter-Mediated Glutamate Secretion in the Mammalian Pineal Gland J. Neurosci., October 22, 2008; 28(43): 10852 - 10863. [Abstract] [Full Text] [PDF]
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H. Misonou, S. M. Thompson, and X. Cai Dynamic Regulation of the Kv2.1 Voltage-Gated Potassium Channel during Brain Ischemia through Neuroglial Interaction J. Neurosci., August 20, 2008; 28(34): 8529 - 8538. [Abstract] [Full Text] [PDF]
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 S. Colleoni, A. A. Jensen, E. Landucci, E. Fumagalli, P. Conti, A. Pinto, M. De Amici, D. E. Pellegrini-Giampietro, C. De Micheli, T. Mennini, et al. Neuroprotective Effects of the Novel Glutamate Transporter Inhibitor (-)-3-Hydroxy-4,5,6,6a-tetrahydro-3aH-pyrrolo[3,4-d]-isoxazole-4-carboxylic Acid, Which Preferentially Inhibits Reverse Transport (Glutamate Release) Compared with Glutamate Reuptake J. Pharmacol. Exp. Ther., August 1, 2008; 326(2): 646 - 656. [Abstract] [Full Text] [PDF]
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 D. Torres-Salazar and C. Fahlke Neuronal Glutamate Transporters Vary in Substrate Transport Rate but Not in Unitary Anion Channel Conductance J. Biol. Chem., November 30, 2007; 282(48): 34719 - 34726. [Abstract] [Full Text] [PDF]
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 Z. Zhang, Z. Tao, A. Gameiro, S. Barcelona, S. Braams, T. Rauen, and C. Grewer Transport direction determines the kinetics of substrate transport by the glutamate transporter EAAC1 PNAS, November 13, 2007; 104(46): 18025 - 18030. [Abstract] [Full Text] [PDF]
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C. Piochon, T. Irinopoulou, D. Brusciano, Y. Bailly, J. Mariani, and C. Levenes NMDA Receptor Contribution to the Climbing Fiber Response in the Adult Mouse Purkinje Cell J. Neurosci., October 3, 2007; 27(40): 10797 - 10809. [Abstract] [Full Text] [PDF]
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A. A. Cattani, V. D. Bonfardin, A. Represa, Y. Ben-Ari, and L. Aniksztejn Generation of Slow Network Oscillations in the Developing Rat Hippocampus After Blockade of Glutamate Uptake J Neurophysiol, October 1, 2007; 98(4): 2324 - 2336. [Abstract] [Full Text] [PDF]
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 Y. Ben-Ari, J.-L. Gaiarsa, R. Tyzio, and R. Khazipov GABA: A Pioneer Transmitter That Excites Immature Neurons and Generates Primitive Oscillations Physiol Rev, October 1, 2007; 87(4): 1215 - 1284. [Abstract] [Full Text] [PDF]
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 S. Teichman and B. I. Kanner Aspartate-444 Is Essential for Productive Substrate Interactions in a Neuronal Glutamate Transporter J. Gen. Physiol., June 1, 2007; 129(6): 527 - 539. [Abstract] [Full Text] [PDF]
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K. L. Meur, M. Galante, M. C. Angulo, and E. Audinat Tonic activation of NMDA receptors by ambient glutamate of non-synaptic origin in the rat hippocampus J. Physiol., April 15, 2007; 580(2): 373 - 383. [Abstract] [Full Text] [PDF]
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Z. Tao and C. Grewer Cooperation of the Conserved Aspartate 439 and Bound Amino Acid Substrate Is Important for High-Affinity Na+ Binding to the Glutamate Transporter EAAC1 J. Gen. Physiol., March 26, 2007; 129(4): 331 - 344. [Abstract] [Full Text] [PDF]
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K. Kaneda, T. Kita, and H. Kita Repetitive Activation of Glutamatergic Inputs Evokes a Long-Lasting Excitation in Rat Globus Pallidus Neurons In Vitro J Neurophysiol, January 1, 2007; 97(1): 121 - 133. [Abstract] [Full Text] [PDF]
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 K. Shimamoto, Y. Otsubo, Y. Shigeri, Y. Yasuda-Kamatani, M. Satoh, S. Kaneko, and T. Nakagawa Characterization of the Tritium-Labeled Analog of L-threo-beta-Benzyloxyaspartate Binding to Glutamate Transporters Mol. Pharmacol., January 1, 2007; 71(1): 294 - 302. [Abstract] [Full Text] [PDF]
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S. G. Owe, P. Marcaggi, and D. Attwell The ionic stoichiometry of the GLAST glutamate transporter in salamander retinal glia J. Physiol., December 1, 2006; 577(2): 591 - 599. [Abstract] [Full Text] [PDF]
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J. D. Pita-Almenar, M. S. Collado, C. M. Colbert, and A. Eskin Different Mechanisms Exist for the Plasticity of Glutamate Reuptake during Early Long-Term Potentiation (LTP) and Late LTP J. Neurosci., October 11, 2006; 26(41): 10461 - 10471. [Abstract] [Full Text] [PDF]
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J. I. Matsui, A. L. Egana, T. R. Sponholtz, A. R. Adolph, and J. E. Dowling Effects of ethanol on photoreceptors and visual function in developing zebrafish. Invest. Ophthalmol. Vis. Sci., October 1, 2006; 47(10): 4589 - 4597. [Abstract] [Full Text] [PDF]
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 S. Cechova and Z. Zuo Inhibition of glutamate transporters increases the minimum alveolar concentration for isoflurane in rats Br. J. Anaesth., August 1, 2006; 97(2): 192 - 195. [Abstract] [Full Text] [PDF]
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E. Glowatzki, N. Cheng, H. Hiel, E. Yi, K. Tanaka, G. C. R. Ellis-Davies, J. D. Rothstein, and D. E. Bergles The glutamate-aspartate transporter GLAST mediates glutamate uptake at inner hair cell afferent synapses in the mammalian cochlea. J. Neurosci., July 19, 2006; 26(29): 7659 - 7664. [Abstract] [Full Text] [PDF]
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D. Torres-Salazar and C. Fahlke Intersubunit interactions in EAAT4 glutamate transporters. J. Neurosci., July 12, 2006; 26(28): 7513 - 7522. [Abstract] [Full Text] [PDF]
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Y. Takayasu, M. Iino, K. Shimamoto, K. Tanaka, and S. Ozawa Glial glutamate transporters maintain one-to-one relationship at the climbing fiber-Purkinje cell synapse by preventing glutamate spillover. J. Neurosci., June 14, 2006; 26(24): 6563 - 6572. [Abstract] [Full Text] [PDF]
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Z. Tao, Z. Zhang, and C. Grewer Neutralization of the Aspartic Acid Residue Asp-367, but Not Asp-454, Inhibits Binding of Na+ to the Glutamate-free Form and Cycling of the Glutamate Transporter EAAC1 J. Biol. Chem., April 14, 2006; 281(15): 10263 - 10272. [Abstract] [Full Text] [PDF]
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S. Satake, S.-Y. Song, Q. Cao, H. Satoh, D. A. Rusakov, Y. Yanagawa, E.-A. Ling, K. Imoto, and S. Konishi Characterization of AMPA receptors targeted by the climbing fiber transmitter mediating presynaptic inhibition of GABAergic transmission at cerebellar interneuron-Purkinje cell synapses. J. Neurosci., February 22, 2006; 26(8): 2278 - 2289. [Abstract] [Full Text] [PDF]
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T. Fellin, T. Pozzan, and G. Carmignoto Purinergic Receptors Mediate Two Distinct Glutamate Release Pathways in Hippocampal Astrocytes J. Biol. Chem., February 17, 2006; 281(7): 4274 - 4284. [Abstract] [Full Text] [PDF]
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J. C. Holt, J.-T. Xue, A. M. Brichta, and J. M. Goldberg Transmission Between Type II Hair Cells and Bouton Afferents in the Turtle Posterior Crista J Neurophysiol, January 1, 2006; 95(1): 428 - 452. [Abstract] [Full Text] [PDF]
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J. Dunlop, H. B. McIlvain, T. A. Carrick, B. Jow, Q. Lu, D. Kowal, S. Lin, A. Greenfield, C. Grosanu, K. Fan, et al. Characterization of Novel Aryl-Ether, Biaryl, and Fluorene Aspartic Acid and Diaminopropionic Acid Analogs as Potent Inhibitors of the High-Affinity Glutamate Transporter EAAT2 Mol. Pharmacol., October 1, 2005; 68(4): 974 - 982. [Abstract] [Full Text] [PDF]
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Y. Takayasu, M. Iino, W. Kakegawa, H. Maeno, K. Watase, K. Wada, D. Yanagihara, T. Miyazaki, O. Komine, M. Watanabe, et al. Differential Roles of Glial and Neuronal Glutamate Transporters in Purkinje Cell Synapses J. Neurosci., September 21, 2005; 25(38): 8788 - 8793. [Abstract] [Full Text] [PDF]
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R. Renden, H. Taschenberger, N. Puente, D. A. Rusakov, R. Duvoisin, L.-Y. Wang, K. P. Lehre, and H. von Gersdorff Glutamate Transporter Studies Reveal the Pruning of Metabotropic Glutamate Receptors and Absence of AMPA Receptor Desensitization at Mature Calyx of Held Synapses J. Neurosci., September 14, 2005; 25(37): 8482 - 8497. [Abstract] [Full Text] [PDF]
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R. I. Melendez, J. Vuthiganon, and P. W. Kalivas Regulation of Extracellular Glutamate in the Prefrontal Cortex: Focus on the Cystine Glutamate Exchanger and Group I Metabotropic Glutamate Receptors J. Pharmacol. Exp. Ther., July 1, 2005; 314(1): 139 - 147. [Abstract] [Full Text] [PDF]
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N. J. Bannister, T. A. Benke, J. Mellor, H. Scott, E. Gurdal, J. W. Crabtree, and J. T. R. Isaac Developmental Changes in AMPA and Kainate Receptor-Mediated Quantal Transmission at Thalamocortical Synapses in the Barrel Cortex J. Neurosci., May 25, 2005; 25(21): 5259 - 5271. [Abstract] [Full Text] [PDF]
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P. Cavelier and D. Attwell Tonic release of glutamate by a DIDS-sensitive mechanism in rat hippocampal slices J. Physiol., April 15, 2005; 564(2): 397 - 410. [Abstract] [Full Text] [PDF]
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J. S. Diamond Deriving the Glutamate Clearance Time Course from Transporter Currents in CA1 Hippocampal Astrocytes: Transmitter Uptake Gets Faster during Development J. Neurosci., March 16, 2005; 25(11): 2906 - 2916. [Abstract] [Full Text] [PDF]
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M. T. Besson, D. B. Re, M. Moulin, and S. Birman High Affinity Transport of Taurine by the Drosophila Aspartate Transporter dEAAT2 J. Biol. Chem., February 25, 2005; 280(8): 6621 - 6626. [Abstract] [Full Text] [PDF]
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K. Y. Wong, A. R. Adolph, and J. E. Dowling Retinal Bipolar Cell Input Mechanisms in Giant Danio. I. Electroretinographic Analysis J Neurophysiol, January 1, 2005; 93(1): 84 - 93. [Abstract] [Full Text] [PDF]
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P. V. Massey, B. E. Johnson, P. R. Moult, Y. P. Auberson, M. W. Brown, E. Molnar, G. L. Collingridge, and Z. I. Bashir Differential Roles of NR2A and NR2B-Containing NMDA Receptors in Cortical Long-Term Potentiation and Long-Term Depression J. Neurosci., September 8, 2004; 24(36): 7821 - 7828. [Abstract] [Full Text] [PDF]
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M. Funicello, P. Conti, M. De Amici, C. De Micheli, T. Mennini, and M. Gobbi Dissociation of [3H]L-Glutamate Uptake from L-Glutamate-Induced [3H]D-Aspartate release by 3-Hydroxy-4,5,6,6a-tetrahydro-3aH-pyrrolo[3,4-d]isoxazole-4-carboxylic Acid and 3-Hydroxy-4,5,6,6a-tetrahydro-3aH-pyrrolo[3,4-d]isoxazole-6-carboxylic Acid, Two Conformationally Constrained Aspartate and Glutamate Analogs Mol. Pharmacol., September 1, 2004; 66(3): 522 - 529. [Abstract] [Full Text] [PDF]
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H. Huang and A. Bordey Glial Glutamate Transporters Limit Spillover Activation of Presynaptic NMDA Receptors and Influence Synaptic Inhibition of Purkinje Neurons J. Neurosci., June 23, 2004; 24(25): 5659 - 5669. [Abstract] [Full Text] [PDF]
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C. Grewer and E. Grabsch New inhibitors for the neutral amino acid transporter ASCT2 reveal its Na+-dependent anion leak J. Physiol., June 15, 2004; 557(3): 747 - 759. [Abstract] [Full Text] [PDF]
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Y. H. Huang, S. R. Sinha, K. Tanaka, J. D. Rothstein, and D. E. Bergles Astrocyte Glutamate Transporters Regulate Metabotropic Glutamate Receptor-Mediated Excitation of Hippocampal Interneurons J. Neurosci., May 12, 2004; 24(19): 4551 - 4559. [Abstract] [Full Text] [PDF]
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G. Brasnjo and T. S. Otis Isolation of glutamate transport-coupled charge flux and estimation of glutamate uptake at the climbing fiber-Purkinje cell synapse PNAS, April 20, 2004; 101(16): 6273 - 6278. [Abstract] [Full Text] [PDF]
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K. Shimamoto, R. Sakai, K. Takaoka, N. Yumoto, T. Nakajima, S. G. Amara, and Y. Shigeri Characterization of Novel L-threo-{beta}-Benzyloxyaspartate Derivatives, Potent Blockers of the Glutamate Transporters Mol. Pharmacol., April 1, 2004; 65(4): 1008 - 1015. [Abstract] [Full Text]
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M. Demarque, N. Villeneuve, J.-B. Manent, H. Becq, A. Represa, Y. Ben-Ari, and L. Aniksztejn Glutamate Transporters Prevent the Generation of Seizures in the Developing Rat Neocortex J. Neurosci., March 31, 2004; 24(13): 3289 - 3294. [Abstract] [Full Text] [PDF]
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H. P. Larsson, A. V. Tzingounis, H. P. Koch, and M. P. Kavanaugh Fluorometric measurements of conformational changes in glutamate transporters PNAS, March 16, 2004; 101(11): 3951 - 3956. [Abstract] [Full Text] [PDF]
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Y. H. Huang, M. Dykes-Hoberg, K. Tanaka, J. D. Rothstein, and D. E. Bergles Climbing Fiber Activation of EAAT4 Transporters and Kainate Receptors in Cerebellar Purkinje Cells J. Neurosci., January 7, 2004; 24(1): 103 - 111. [Abstract] [Full Text] [PDF]
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H. Zhang and D. Sulzer Glutamate Spillover in the Striatum Depresses Dopaminergic Transmission by Activating Group I Metabotropic Glutamate Receptors J. Neurosci., November 19, 2003; 23(33): 10585 - 10592. [Abstract] [Full Text] [PDF]
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P. Marcaggi, D. Billups, and D. Attwell The Role of Glial Glutamate Transporters in Maintaining the Independent Operation of Juvenile Mouse Cerebellar Parallel Fibre Synapses J. Physiol., October 1, 2003; 552(1): 89 - 107. [Abstract] [Full Text] [PDF]
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P. Aoun, J. W. Simpkins, and N. Agarwal Role of PPAR-{gamma} Ligands In Neuroprotection against Glutamate-Induced Cytotoxicity in Retinal Ganglion Cells Invest. Ophthalmol. Vis. Sci., July 1, 2003; 44(7): 2999 - 3004. [Abstract] [Full Text] [PDF]
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M. J. Palmer, H. Taschenberger, C. Hull, L. Tremere, and H. von Gersdorff Synaptic Activation of Presynaptic Glutamate Transporter Currents in Nerve Terminals J. Neurosci., June 15, 2003; 23(12): 4831 - 4841. [Abstract] [Full Text] [PDF]
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M. Tanaka, N. Maeda, M. Noda, and T. Marunouchi A Chondroitin Sulfate Proteoglycan PTPzeta /RPTPbeta Regulates the Morphogenesis of Purkinje Cell Dendrites in the Developing Cerebellum J. Neurosci., April 1, 2003; 23(7): 2804 - 2814. [Abstract] [Full Text] [PDF]
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G. C. Mathews and J. S. Diamond Neuronal Glutamate Uptake Contributes to GABA Synthesis and Inhibitory Synaptic Strength J. Neurosci., March 15, 2003; 23(6): 2040 - 2048. [Abstract] [Full Text] [PDF]
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D. E. Bergles, A. V. Tzingounis, and C. E. Jahr Comparison of Coupled and Uncoupled Currents during Glutamate Uptake by GLT-1 Transporters J. Neurosci., December 1, 2002; 22(23): 10153 - 10162. [Abstract] [Full Text] [PDF]
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D. A. Baker, Z.-X. Xi, H. Shen, C. J. Swanson, and P. W. Kalivas The Origin and Neuronal Function of In Vivo Nonsynaptic Glutamate J. Neurosci., October 15, 2002; 22(20): 9134 - 9141. [Abstract] [Full Text] [PDF]
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B. H. Leighton, R. P. Seal, K. Shimamoto, and S. G. Amara A Hydrophobic Domain in Glutamate Transporters Forms an Extracellular Helix Associated with the Permeation Pathway for Substrates J. Biol. Chem., August 9, 2002; 277(33): 29847 - 29855. [Abstract] [Full Text] [PDF]
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N. MacAulay, U. Gether, D. A Klaerke, and T. Zeuthen Passive water and urea permeability of a human Na+-glutamate cotransporter expressed in Xenopus oocytes J. Physiol., August 1, 2002; 542(3): 817 - 828. [Abstract] [Full Text] [PDF]
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B. A. Clark and S. G. Cull-Candy Activity-Dependent Recruitment of Extrasynaptic NMDA Receptor Activation at an AMPA Receptor-Only Synapse J. Neurosci., June 1, 2002; 22(11): 4428 - 4436. [Abstract] [Full Text] [PDF]
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R. M. Ryan and R. J. Vandenberg Distinct Conformational States Mediate the Transport and Anion Channel Properties of the Glutamate Transporter EAAT-1 J. Biol. Chem., April 12, 2002; 277(16): 13494 - 13500. [Abstract] [Full Text] [PDF]
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S. Chen and J. S. Diamond Synaptically Released Glutamate Activates Extrasynaptic NMDA Receptors on Cells in the Ganglion Cell Layer of Rat Retina J. Neurosci., March 15, 2002; 22(6): 2165 - 2173. [Abstract] [Full Text] [PDF]
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T. Numakawa, S. Yamagishi, N. Adachi, T. Matsumoto, D. Yokomaku, M. Yamada, and H. Hatanaka Brain-derived Neurotrophic Factor-induced Potentiation of Ca2+ Oscillations in Developing Cortical Neurons J. Biol. Chem., February 15, 2002; 277(8): 6520 - 6529. [Abstract] [Full Text] [PDF]
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Z.-X. Xi, D. A. Baker, H. Shen, D. S. Carson, and P. W. Kalivas Group II Metabotropic Glutamate Receptors Modulate Extracellular Glutamate in the Nucleus Accumbens J. Pharmacol. Exp. Ther., January 1, 2002; 300(1): 162 - 171. [Abstract] [Full Text] [PDF]
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R. P. Seal, Y. Shigeri, S. Eliasof, B. H. Leighton, and S. G. Amara Sulfhydryl modification of V449C in the glutamate transporter EAAT1 abolishes substrate transport but not the substrate-gated anion conductance PNAS, December 18, 2001; 98(26): 15324 - 15329. [Abstract] [Full Text] [PDF]
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J. S. Diamond Neuronal Glutamate Transporters Limit Activation of NMDA Receptors by Neurotransmitter Spillover on CA1 Pyramidal Cells J. Neurosci., November 1, 2001; 21(21): 8328 - 8338. [Abstract] [Full Text] [PDF]
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 A. Miyazato, S. Ueno, K. Ohmine, M. Ueda, K. Yoshida, Y. Yamashita, T. Kaneko, M. Mori, K. Kirito, M. Toshima, et al. Identification of myelodysplastic syndrome-specific genes by DNA microarray analysis with purified hematopoietic stem cell fraction Blood, July 15, 2001; 98(2): 422 - 427. [Abstract] [Full Text] [PDF]
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M. V Poulsen and R. J Vandenberg Niflumic acid modulates uncoupled substrate-gated conductances in the human glutamate transporter EAAT4 J. Physiol., July 1, 2001; 534(1): 159 - 167. [Abstract] [Full Text] [PDF]
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R. Sakai, G. T. Swanson, K. Shimamoto, T. Green, A. Contractor, A. Ghetti, Y. Tamura-Horikawa, C. Oiwa, and H. Kamiya Pharmacological Properties of the Potent Epileptogenic Amino Acid Dysiherbaine, a Novel Glutamate Receptor Agonist Isolated from the Marine Sponge Dysidea herbacea J. Pharmacol. Exp. Ther., April 13, 2001; 296(2): 650 - 658. [Abstract] [Full Text]
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C. Grewer, N. Watzke, M. Wiessner, and T. Rauen Glutamate translocation of the neuronal glutamate transporter EAAC1 occurs within milliseconds PNAS, August 6, 2000; (2000) 160170397. [Abstract] [Full Text]
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D. Jabaudon, M. Scanziani, B. H. Gahwiler, and U. Gerber Acute decrease in net glutamate uptake during energy deprivation PNAS, May 9, 2000; 97(10): 5610 - 5615. [Abstract] [Full Text] [PDF]
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H. P. Koch, M. P. Kavanaugh, C. S. Esslinger, N. Zerangue, J. M. Humphrey, S. G. Amara, A. R. Chamberlin, and R. J. Bridges Differentiation of Substrate and Nonsubstrate Inhibitors of the High-Affinity, Sodium-Dependent Glutamate Transporters Mol. Pharmacol., December 1, 1999; 56(6): 1095 - 1104. [Abstract] [Full Text]
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S. Mennerick, W. Shen, W. Xu, A. Benz, K. Tanaka, K. Shimamoto, K. E. Isenberg, J. E. Krause, and C. F. Zorumski Substrate Turnover by Transporters Curtails Synaptic Glutamate Transients J. Neurosci., November 1, 1999; 19(21): 9242 - 9251. [Abstract] [Full Text] [PDF]
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 R. A. Swanson and S. Duan Regulation of Glutamate Transporter Function Neuroscientist, September 1, 1999; 5(5): 280 - 282. [Abstract] [PDF]
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D. Jabaudon, K. Shimamoto, Y. Yasuda-Kamatani, M. Scanziani, B. H. Gahwiler, and U. Gerber Inhibition of uptake unmasks rapid extracellular turnover of glutamate of nonvesicular origin PNAS, July 20, 1999; 96(15): 8733 - 8738. [Abstract] [Full Text] [PDF]
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 D. J. Slotboom, W. N. Konings, and J. S. Lolkema Structural Features of the Glutamate Transporter Family Microbiol. Mol. Biol. Rev., June 1, 1999; 63(2): 293 - 307. [Abstract] [Full Text] [PDF]
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C. Grewer, N. Watzke, M. Wiessner, and T. Rauen Glutamate translocation of the neuronal glutamate transporter EAAC1 occurs within milliseconds PNAS, August 15, 2000; 97(17): 9706 - 9711. [Abstract] [Full Text] [PDF]
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), DL-TBzOAsp (
), DHKA (
), 2S4R4MG (
), DL-THA (
), and
L-CCG-III (
). Specific uptake of
[14C]glutamate were expressed as relative ratio to
control. Values are presented as mean ± standard error of at
least three determinations.
), 2 (
