Characterization of the tritium-labeled analog of L-threo-β- benzyloxyaspartate binding to glutamate transporters

Article abstract of DOI:10.1124/mol.106.027250

L-Glutamate is the major excitatory neurotransmitter in the mammalian central nervous system. Termination of glutamate receptor activation and maintenance of low extracellular glutamate concentrations are primarily achieved by glutamate transporters (exci

Full text of DOI:10.1124/mol.106.027250

0026-895X/07/7101-294–302$20.00
MOLECULAR PHARMACOLOGY
Copyright © 2007 The American Society for Pharmacology and Experimental Therapeutics
Mol Pharmacol 71:294–302, 2007
Vol. 71, No. 1
27250/3164691
Printed in U.S.A.
Characterization of the Tritium-Labeled Analog of L-threo-␤-
Benzyloxyaspartate Binding to Glutamate Transporters
Keiko Shimamoto, Yasuto Otsubo, Yasushi Shigeri, Yoshimi Yasuda-Kamatani,
Masamichi Satoh, Shuji Kaneko, and Takayuki Nakagawa
Suntory Institute for Bioorganic Research, Wakayamadai, Shimamoto-cho, Mishima-gun, Osaka, Japan (K.S., Y.Y.-K.);
Department of Molecular Pharmacology, Graduate School of Pharmaceutical Sciences, Kyoto University, Kyoto, Japan (Y.O.,
S.K., T.N.); Yasuda Women’s University, Hiroshima, Japan (M.S.); and National Institute of Advanced Industrial Science and
Technology, Midorigaoka, Ikeda, Japan (Y.S.)
Received May 29, 2006; accepted October 17, 2006
ABSTRACT
L-Glutamate is the major excitatory neurotransmitter in the and we therefore synthesized a tritiated derivative of (2S,3S)-
mammalian central nervous system. Termination of gluta- 3-{3-[4-ethylbenzoylamino]benzyloxy}aspartate
([3H]ETB-
mate receptor activation and maintenance of low extracellu- TBOA). [3H]ETB-TBOA showed significant high-affinity spe-
lar glutamate concentrations are primarily achieved by glu- cific binding to EAAT-transfected COS-1 cell membranes
tamate transporters (excitatory amino acid transporters 1–5, with each EAAT subtype. The Hill coefficient for the Naϩ-
EAATs1–5) located on both the nerve endings and the sur- dependence of [3H]ETB-TBOA binding revealed a single
rounding glial cells. To identify the physiological roles of class of noncooperative binding sites for Naϩ, suggesting
each subtype, subtype-selective EAAT ligands are required. that Naϩ binding in the ligand binding step is different from
In this study, we developed a binding assay system to char- Naϩ binding in the substrate uptake process. The binding
acterize EAAT ligands for all EAAT subtypes. We recently was displaced by known substrates and blockers. The rank
synthesized novel analogs of threo-␤-benzyloxyaspartate order of inhibition by these compounds was consistent with
(TBOA) and reported that they blocked glutamate uptake by glutamate uptake assay results reported previously. Thus,
EAATs 1–5 much more potently than TBOA. The strong the [3H]ETB-TBOA binding assay will be useful to screen
inhibitory activity of the TBOA analogs suggested that they novel EAAT ligands for all EAAT subtypes.
would be suitable to use as radioisotope-labeled ligands,
Glutamate acts as a major excitatory neurotransmitter in tion at low levels to limit the activation of glutamate recep-
the mammalian central nervous system and has been impli- tors and to protect neurons from excitotoxicity (Danbolt,
cated in higher brain function and in brain pathophysiology.
Excitatory amino acid transporters (EAATs) play important
roles in maintaining the extracellular glutamate concentra-
2001). Five subtypes of EAATs have been identified. EAAT1
(GLAST) is expressed in astrocytes throughout the brain,
including the cerebellum. EAAT2 (GLT-1) is the most prev-
alent subtype and is expressed primarily in astrocytes in the
forebrain. These two glial transporters are responsible for
the majority of glutamate uptake and are critical to the
maintenance of glutamate homeostasis in brain. EAAT3
(EAAC1) and EAAT4 are found at postsynaptic nerve end-
ings. EAAT3 is widely distributed in the brain, whereas
EAAT4 specifically localizes to the cerebellum (Purkinje
cells). EAAT5 is a photoreceptor and bipolar cell glutamate
transporter selectively expressed in the retina.
This work was supported in part by Grants-in-Aid for Creative Scientific
Research 13NP0401 (to K.S.) and the Special Coordination Funds for Promot-
ing Science and Technology Target-Oriented Brain Science Research Program
(to T.N.), Grants-in-Aid for Scientific Research 15500278 (to Y.S.), 16510174
(to K.S.), 18613006 (to T.N.), and
a Grant-in-Aid for Young Scientists
16790051 (to T.N.) from the Ministry of Education, Science, Sports, and Cul-
ture of Japan.
K.S. and T.N. contributed equally to this work.
Article, publication date, and citation information can be found at
http://molpharm.aspetjournals.org.
doi:10.1124/mol.106.027250.
The uptake of glutamate by EAATs is driven by electro-
ABBREVIATIONS: EAAT, excitatory amino acid transporter; TBOA, threo-␤-benzyloxyaspartate; ETB-TBOA, (2S,3S)-3-{3-[4-ethylben-
zoylamino]benzyloxy}aspartate; TFB-TBOA, (2S,3S)-3-{3-[4-(trifluoromethyl)benzoylamino]benzyloxy}aspartate; PMB-TBOA, (2S,3S)-3-{3-
[4-methoxybenzoylamino]benzyloxy}aspartate; CCG-II, (2S,1ЈR,2ЈR)-2-(2-carboxycyclopropyl)glycine; CCG-III, (2S,1ЈS,2ЈR)-2-(2-carboxycyclo-
propyl)glycine; iGluR, ionotropic glutamate receptor; mGluR, metabotropic glutamate receptor; Vin-BzA-TBOA, (2S,3S)-3-{3-[4-vinyl-
benzoylamino]benzyloxy}aspartate; HPLC, high-performance liquid chromatography; TLC, thin layer chromatography; PBS, phosphate-buffered
saline; L-TBOA, (2S,3S)-3-benzyloxyaspartate; DHKA, dihydrokainate; [3H]4MG, [3H](2S,4R)-4-methylglutamate.
294

Glutamate Transporter Binding Assay
295
chemical gradients across the plasma membrane. The pro- noncooperative binding sites for Naϩ, suggesting that Naϩ
cess also requires Naϩ for substrate binding and Kϩ for net binding in the ligand binding step is different from Naϩ
transport. It has been proposed that three Naϩ ions and one binding in the substrate uptake process. The binding of [3H-
Hϩ are transported into the cell with glutamate, whereas one ]ETB-TBOA was displaced by known substrate-type inhibi-
Kϩ ion is transported out (Zerangue and Kavanaugh, 1996; tors [L-glutamate, L-aspartate, (2S,1ЈS,2ЈR)-2-(2-carboxycy-
Levy et al., 1998). In addition, an anion conductance that is clopropyl)glycine (CCG-III), etc.] and blocker-type inhibitors
thermodynamically uncoupled to glutamate transport has (TBOA, TFB-TBOA, etc.). The rank order of inhibition by
been demonstrated (Fairman et al., 1995; Wadiche et al., these compounds was consistent with results reported
1995; Arriza et al., 1997), although this property varies previously in the glutamate uptake assay and in elec-
among the EAAT subtypes. EAAT2 does not exhibit this trophysiological studies. This binding assay also enabled us
property, but a ClϪ conductance representing more than 95% to quantify the responses of EAAT4 and EAAT5. These
of the observed steady-state current associated with gluta- findings suggest that [3H]ETB-TBOA will serve as a useful
mate uptake is associated with EAAT4 and EAAT5 (Bergles tool to screen novel EAAT ligands for all EAAT subtypes.
et al., 2002).
Although important roles of EAATs have been elucidated
using genetic approaches, genetic manipulations cannot pro-
vide information on the dynamics of glutamate homeostasis
in response to short-term disruption of uptake systems.
Thus, pharmacological intervention using blockers is an in-
dispensable approach for investigating the physiological
roles of each subtype (Campiani et al., 2003; Shigeri et al.,
2004; Bridges and Esslinger, 2005; Shimamoto and Shigeri,
Materials and Methods
Materials. ETB-TBOA and (2S,3S)-3-{3-[4-vinylbenzoylamino]-
benzyloxy}aspartate (Vin-BzA-TBOA) were synthesized as optically
pure forms from a commercially available epoxide, (2S,3R)-[3-(ben-
zyloxymethyl)oxiranyl]methanol para-nitorobenzoate (Fluka Che-
mie AG, Buchs, Switzerland) in the same manner as TFB-TBOA
(Shimamoto et al., 2004). The structure and purity (Ͼ95%) of the
compounds were confirmed by 400 MHz NMR and HPLC. Stock
solutions (10 mM) were prepared in dimethyl sulfoxide as trifluoro-
2006). We have demonstrated that (2S,3S)-3-benzyloxyas-
partate (L-TBOA) is nontransportable for all subtypes of
acetic acid salts and were stored at Ϫ20°C until the day of the
EAATs (Shimamoto et al., 1998; Shigeri et al., 2001). Re- experiments. TBOA analogs were confirmed to be stable in the stock
solutions for more than 3 months. Tritium-labeled ETB-TBOA
([3H]ETB-TBOA) was prepared by Daiichi Pure Chemicals Co., Ltd.
(Tokyo, Japan), as shown in Fig. 1. The specific radioactivity was
1.48 TBq/mmol, and the concentration was 37.0 MBq/ml. Chemical
and radiochemical purities were confirmed to be Ͼ97% by TLC and
HPLC. TLC (Silica gel 60 F254; Merck, Darmstadt, Germany) was
developed in 1-butanol/acetic acid/water at a ratio of 4:1:2 (v/v), and
radioactivity was detected using a bioimaging analyzer (BAS2000;
Fuji Photo Film, Tokyo, Japan). HPLC was performed using a Cos-
mosil 5 C18-MSII column (4.6-mm i.d.ϫ 150 mm; Nacalai Tesque,
cently, we synthesized novel TBOA analogs and reported
that benzoylamide derivatives of TBOA [(2S,3S)-3-{3-[4-
substituted-benzoylamino]benzyloxy}aspartate]
blocked
EAATs1–5 much more potently than TBOA in electrophysi-
ological assays using EAATs expressed in Xenopus laevis
oocytes (Shimamoto et al., 2004). We also found that low
nanomolar concentrations of these compounds inhibited glu-
tamate uptake by COS-1 cells expressing EAATs1–3. How-
ever, we could not evaluate glutamate uptake by EAAT4 or
EAAT5 because the glutamate uptake capacities of these Kyoto, Japan), and the retention time was 5.40 min [eluent: aceto-
nitrile/water 3:7 (v/v) containing 0.1% trifluoroacetic acid; flow rate:
1.0 ml/min; detection: UV (254 nm)]. The radioactivity of each peak
was detected by adding the scintillation cocktail FLO-SCINT II
(PerkinElmer Life and Analytical Sciences, Boston, MA). [3H]ETB-
TBOA was stored in ethanol at Ϫ20°C. Other radiolabeled ligands
were purchased from PerkinElmer. All other chemicals were of the
highest purity available and were purchased from Nacalai Tesque,
Sigma (St. Louis, MO), or Tocris Cookson (Bristol, UK).
subtypes are much lower than those of EAATs1–3. Moreover,
irreversible inhibition by the TBOA derivatives prevented
quantitative analysis in the electrophysiological assay. Thus,
we could not evaluate the detailed effects of these compounds
on EAAT4 or EAAT5. Likewise, only a few inhibitors of
EAAT4 and EAAT5 have been characterized in other reports
(Fairman et al., 1995; Arriza et al., 1997; Eliasof et al., 2001;
Shigeri et al., 2001), whereas most pharmacological studies
to develop subtype-selective blockers have only focused on
Transfection. Eukaryotic expression vectors (pKDEMSS) con-
taining cDNA of EAATs1–3 were prepared as reported previously
EAATs1–3. Thus, a simple assay system in which the effects (Shimamoto et al., 1998). EAAT4 cDNA was obtained from a human
cerebellum cDNA library (Clontech, Palo Alto, CA) by polymerase
chain reaction using a 5Ј-upstream primer (5Ј-ATAGACCATGAG-
CAGCCATGGCA-3Ј) and 3Ј-downstream primer (5Ј-CTCACATAG-
CACTCTCGTTGCCT-3Ј). The products were cloned into pCRII (In-
of compounds can be quantitatively compared among all
EAAT subtypes would be useful for evaluating new com-
pounds and for screening subtype-selective EAAT ligands.
In the present study, we developed a novel binding assay
system for EAATs. The TBOA analogs are potent inhibitors
of EAATs, making them highly suitable for use as radioiso-
tope-labeled ligands. Therefore, we synthesized a tritium
labeled analog of (2S,3S)-3-{3-[4-ethylbenzoylamino]-
benzyloxy}aspartate ([3H]ETB-TBOA). The potency of ETB-
TBOA inhibition of EAATs1–3 in the glutamate uptake assay
was comparable with that of (2S,3S)-3-{3-[4-(trifluoro-
methyl)benzoylamino]benzyloxy}aspartate (TFB-TBOA), the
most potent TBOA analog. [3H]ETB-TBOA showed specific
binding to rat brain crude membranes and EAAT-transfected
COS-1 cell membranes and bound all of the EAAT subtypes
with high affinity. The Hill coefficient for the Naϩ-depend-
ence of [3H]ETB-TBOA binding revealed a single class of
Fig. 1. Synthesis of [3H]ETB-TBOA from Vin-BzA-TBOA.

296
Shimamoto et al.
vitrogen, Carlsbad, CA) and analyzed by DNA sequencing. A clone Na2HPO4, 1.5 mM KH2PO4, 1 mM MgCl2, 1 mM CaCl2, and 10 mM
containing EAAT4 was digested with EcoRI and subcloned into the D-glucose, pH 7.4, and preincubated in 300 ␮l of the same buffer at
EcoRI site of pKDEMSS. EAAT5 cDNA was obtained using the 37°C for 12 min. After aspiration of the buffer, cells were incubated
following primers for polymerase chain reaction with pOTV-EAAT5 with 1 ␮M L-[14C]glutamate in 100 ␮l of the modified PBS in the
as a template: 5Ј-upstream primer (5Ј-GGAATTCCACCATGGTGC- presence or absence of test compounds at various concentrations at
CGCATACCATC-3Ј) and 3Ј-downstream primer (5Ј-GACTAGTCT- 37°C for 12 min. To terminate uptake, cells were washed three times
CAGACATTGGTCTCCAGCTC-3Ј). The EcoRI (underlined)-SpeI with ice-cold buffer. Radioactivity was measured by scintillation
counting (TopCount; PerkinElmer) in 100 ␮l of MicroScint 40
(PerkinElmer). Nonspecific uptake was determined in the presence
of 1 ␮M TFB-TBOA. Specific uptake of [14C]glutamate is presented
relative to the control. All values displayed are the mean Ϯ S.E.M. of
at least three experiments.
(bold and underlined) fragment was inserted into the EcoRI-SpeI site
of pKDEMSS and analyzed by DNA sequencing. COS-1 cells were
cultured in Dulbecco’s modified Eagle’s medium containing 10% fetal
bovine serum at 37°C in an atmosphere of 5% CO2. Cells were
transfected by electroporation (1 ϫ 107 cells, 200 V, 975 ␮F) with 10
␮g of the vector containing each EAAT subtype. COS-1 cells trans-
fected by the vector alone were used as the control. For the binding
assay, transfected cells were cultured for 2 days, harvested using
EDTA-trypsin, washed with phosphate-buffered saline (PBS), and
stored at Ϫ80°C until use.
Membrane Preparation. On the day of the assay, the frozen
cells were suspended in binding buffer (120 mM NaCl, 5 mM KCl, 2.5
mM CaCl2, 1 mM MgCl2, and 50 mM Tris-HCl, pH 7.6) using a
Polytron homogenizer (Kinematica, Basel, Switzerland) (setting 6,
30 s), followed by centrifugation at 20,000g for 10 min. The pellet was
resuspended in the binding buffer at a final concentration of 100 to
500 ␮g/ml. To examine ion dependence, several buffers were pre-
pared as follows. NaCl buffer contained 120 mM NaCl, 5 mM KCl,
2.5 mM CaCl2, and 1 mM MgCl2. To examine the concentration
response for Naϩ, various concentrations of NaCl were replaced by
the same concentration of choline chloride. In LiCl buffer, ChCl
buffer, and KCl buffer, 120 mM LiCl, choline chloride, or KCl, re-
spectively, were substituted for NaCl in the NaCl buffer. For chlo-
ride-free buffer, all ClϪ ions in the NaCl buffer were replaced by
gluconate, and for potassium-free buffer, KCl in the NaCl buffer was
replaced with NaCl. Cell pellets were washed twice with the specific
buffer used in the assay before resuspension in the same buffer at the
final assay concentration. Rat brain crude membranes were pre-
pared from forebrain as described previously (Murphy et al., 1987;
Sakai et al., 2001) and stored at Ϫ80°C until use. The membranes
were washed with the binding buffer and resuspended at a final
concentration of 50 to 100 ␮g/ml.
Results
Synthesis of Tritiated Ligand. In recent years, many
groups have reported novel inhibitors for EAATs. However,
interactions with all EAAT subtypes have only been charac-
terized for a few of these compounds. For most compounds,
only inhibition of EAATs1–3 has been characterized because
glutamate uptake by EAAT4 and EAAT5 is not of sufficient
magnitude to be evaluated experimentally. We compared
[
14C]glutamate uptake by COS-1 cells expressing EAATs1–5.
The amount of [14C]glutamate uptake by EAATs1–5 was
5.3 Ϯ 0.3-, 5.8 Ϯ 0.4-, 7.5 Ϯ 0.3-, 2.1 Ϯ 0.1-, and 1.5 Ϯ 0.2-fold
relative to the control (vector-transfected cells), respectively.
Because COS-1 cells express endogenous transporters, eval-
uation of specific uptake by EAAT4 or EAAT5 in the uptake
assay was not possible. Thus, we have developed a novel
binding assay system capable of evaluating all EAAT sub-
types. Recently, we demonstrated that low nanomolar con-
centrations of TBOA analogs inhibited glutamate uptake and
blocked glutamate-induced transport currents in X. laevis
oocytes much more potently than TBOA (Shimamoto et al.,
2004), without any effects on other receptors and transport-
ers. We synthesized several TBOA analogs as candidates for
a radioligand and found that [3H]ETB-TBOA can be easily
labeled from the vinyl precursor (Vin-BzA-TBOA) by tritium
gas addition in the presence of a palladium catalyst (Fig. 1).
Chemical and radiochemical purity of the compound was
confirmed to be Ͼ97% by silica gel TLC and by HPLC. The
IC50 values of unlabeled ETB-TBOA in the [14C]glutamate
uptake assay using MDCK cells stably expressing EAAT2 or
EAAT3 were 3.2 Ϯ 0.3 and 88 Ϯ 4.8 nM, respectively. These
values are very similar to IC50 values of TFB-TBOA, which
is, thus far, the most potent blocker of these transporters
[1.9 Ϯ 0.1 nM (EAAT2) and 28 Ϯ 1.8 nM (EAAT3)] (Shi-
mamoto et al., 2004). ETB-TBOA did not show any signifi-
cant activity in several other assays, including a binding
assay for ionotropic glutamate receptors (iGluRs) (␣-amino-
3-hydroxy-5-methyl-4-isoxazolepropionic acid, kainate, and
N-methyl-D-aspartate subtypes), a Ca2ϩ mobilization assay
Transporter Binding Assay Using Cell Membranes. An ali-
quot of membranes (200 ␮l) was incubated with the designated
concentration of [3H]ETB-TBOA in the presence or absence of vari-
ous concentrations of test compounds, in triplicate, for 30 min at
room temperature. Nonspecific binding was determined in the pres-
ence of 10 ␮M TFB-TBOA. Bound radioactivity was separated from
free radioactivity by filtration through a glass filter (Filtermat A;
PerkinElmer) using a Micro Cell Harvester (Skatron Instruments
AS, Tranby, Norway). The filter was attached to a solid scintillation
cocktail (MeltiLexA; PerkinElmer) and counted using
a Beta-
plate1205 (PerkinElmer). A Kd value for each EAAT subtype was
obtained from a Scatchard plot of the data. Dose-response curves
were fitted to the Hill equation using Prism III (GraphPad Software
Inc., San Diego, CA). IC50 values were obtained from the dose-
response curves and presented as mean Ϯ S.E.M. from at least three
experiments. Ki values were determined from the equation Ki
IC50/(1 ϩ [L]/Kd), where [L] is the concentration of [3H]ETB-TBOA.
For saturation experiments, binding of [3H]ETB-TBOA was mea- for metabotropic glutamate receptors (mGluR1 and
ϭ
sured over concentrations range of 5 to 100 nM for EAAT1,2,4,5 and
10–300 nM for EAAT3. To generate a Naϩ concentration-response
curve, 20 nM [3H]ETB-TBOA was used, and the specific binding at
each Naϩ concentration was normalized to the amount bound at 120
mM Naϩ (100%). The Hill coefficient was determined from the slope
of a Hill plot of log[B/(Bmax Ϫ B)] as a function of log[Naϩ].
mGluR5), a cAMP formation inhibition assay (mGluR2 and
mGluR4), or a glutamate uptake assay using isolated synap-
tic vesicles (data not shown). HPLC analysis of unlabeled
ETB-TBOA showed that no detectable decomposition oc-
curred after 1 month of storage (10 mM dimethyl sulfoxide
solution), even at room temperature. The transporter binding
characteristics of [3H]ETB-TBOA did not decrease after 1
year of storage in ethanol at Ϫ20°C.
Measurements of [14C]Glutamate Uptake in Transfected
COS-1 Cells. Transfected COS-1 cells or MDCK cells stably express-
ing EAAT2 or EAAT3 were seeded in 96-well plates and cultured for
2 days. The subconfluent cells were washed two times with 300 ␮l of
[3H]ETB-TBOA Binding to Rat Brain Crude Mem-
modified PBS that contained 137 mM NaCl, 2.7 mM KCl, 8.1 mM branes. First, we characterized [3H]ETB-TBOA binding to

Glutamate Transporter Binding Assay
297
rat brain crude membranes, which contain both glial and branes, even at agonist concentrations of 1 mM (data not
neuronal membranes. This preparation is expected to reflect shown).
the characteristics of the endogenously expressed transport-
Saturation Experiments Using EAAT-Expressing
ers and is eminently suitable for the initial screening of novel Cell Membranes. Next, we characterized the binding of
ligands. At a concentration of 10 nM, specific binding was [3H]ETB-TBOA to each EAAT subtype expressed on COS-1
linear with respect to membrane concentration over the cells. These experiments used 30 to 100 ␮g of protein/well. No
range of 5 to 50 ␮g protein/well (data not shown). [3H]ETB- specific binding was observed in membranes prepared from
TBOA rapidly associated with crude membranes (Fig. 2A). vector-transfected cells. However, saturable-specific binding
Equilibrium was reached within 5 min at room temperature, was observed in EAAT-expressing cell membranes (Fig. 4A).
and specific binding was sustained for more than 120 min. Nonspecific binding accounted for approximately 5 to 15% of
Dissociation of more than 90% of bound [3H]ETB-TBOA by total radioactivity at the concentrations used in the displace-
the addition of 10 ␮M TFB-TBOA occurred within 10 min. ment assays (10–20 nM), with the exception of EAAT3-ex-
Although only 30 and 85% of bound radioactivity was disso- pressing cell membranes, in which the specific binding was
ciated after 30 min in the presence of 1 or 10 mM L-gluta- less than the other subtypes, reflecting the low affinity of
mate, respectively, dissociation reached equilibrium within ETB-TBOA for EAAT3. Curve-fitting of saturation experi-
10 min (Fig. 2B). With increasing concentrations of [3H]ETB- ment data and linear Scatchard plots (Fig. 4B) revealed a
TBOA saturable-specific binding was observed with a Kd of one-component binding site for each EAAT subtype, although
29.5 Ϯ 3.7 nM and a Bmax of 41.1 Ϯ 6.8 pmol/mg protein (Fig. the Kd and Bmax values varied (Table 1). The Kd values of
3, A and B). Nonspecific binding, determined by the addition
of 10 ␮M TFB-TBOA, increased linearly and accounted for
approximately 15% of total radioactivity bound at 10 nM
radioligand and approximately 30% at 100 nM. [3H]ETB-
TBOA binding was displaced by EAAT substrates such as
L-glutamate, L-aspartate, and D-aspartate (Fig. 3C). The af-
finity of L-aspartate and D-aspartate was almost comparable,
but that of D-glutamate was lower than L-glutamate. Neither
iGluR agonists [␣-amino-3-hydroxy-5-methyl-4-isoxazolepro-
pionic acid, N-methyl-D-aspartate, and (2S,1ЈR,2ЈS)-2-(2-car-
boxycyclopropyl)glycine] (CCG-IV) nor mGluR agonists
[(1S*,3R*)-1-aminocyclopentane-1,3-dicarboxilic acid and
(2S,2ЈR,3ЈR)-2-(2,3-dicarboxycyclopropyl)glycine] (DCG-IV)
inhibited [3H]ETB-TBOA binding to rat brain crude mem-
Fig. 2. [3H]ETB-TBOA binding to and dissociation from rat brain crude
membranes. A, association of 10 nM [3H]ETB-TBOA at room tempera-
ture. Bound radioactivities were normalized to the value at 60 min. B,
dissociation of 10 nM [3H]ETB-TBOA in the presence of 1 mM L-gluta-
mate (E), 10 mM L-glutamate (Ⅺ), or 10 ␮M TFB-TBOA (Œ) after a
30-min association. Data represent the mean Ϯ S.E.M. from three sepa-
rate experiments.
Fig. 3. [3H]ETB-TBOA binding to rat brain crude membranes. A, repre-
sentative saturation plot; B, its Scatchard analysis. C, inhibition of
[3H]ETB-TBOA binding by EAAT substrates. Nonspecific binding was
determined in the presence of 10 ␮M TFB-TBOA. Data represent mean Ϯ
S.E.M. of at least three separate experiments.

298
Shimamoto et al.
EAAT1,2,4,5 were 10 to 25 nM, whereas that of EAAT3 was [3H]ETB-TBOA binding was investigated using EAAT2-ex-
10-fold higher than the other subtypes. pressing cell membranes. Specific binding completely disap-
Ion Dependence. Because transport of glutamate by peared in ChCl buffer in which NaCl was replaced by choline
EAATs is Naϩ/Kϩ-dependent, the ion dependence of chloride (Fig. 5A). When the Naϩ concentration was in-
Fig. 4. [3H]ETB-TBOA binding to EAAT-expressing COS-1 cell membranes. A, representative saturation plots. Specific binding was detected in
EAAT-expressing cell membranes but not in vector-expressing cell membranes (mock). B, Scatchard analysis of the saturation data.

Glutamate Transporter Binding Assay
299
creased, the specific binding increased, and the Kd values [3H]ETB-TBOA binding was independent of ClϪ and Kϩ ions
decreased. To determine the stoichiometry of Naϩ binding in as shown by binding in chloride-free buffer or potassium-free
the binding process, the Naϩ concentration-dependence of buffer. The uptake of [14C]glutamate was slightly reduced in
[3H]ETB-TBOA binding was examined (Fig. 5B). Specific the absence of ClϪ or Kϩ but not to the extent measured in
binding at each Naϩ concentration was normalized to the Naϩ-free buffers. There were no differences in cell viability
value at 120 mM Naϩ (100%). The Hill coefficient determined in the different buffers during the uptake assay.
from a Hill plot of the data were 1.10 Ϯ 0.03 (r2 ϭ 0.99) and
Displacement Assay. The effects of other EAAT inhibi-
the Km was 53.3 Ϯ 5.5 mM when the dose-response curve was tors were tested on each subtype (Fig. 7). The Ki values for
fitted to a single binding-site model. Other ion dependencies each compound are summarized in Table 2. Both substrate-
were compared between the [3H]ETB-TBOA binding assay type inhibitors (L-trans-pyrrolidine-2,4-dicarboxylic acid and
and the [14C]glutamate uptake assay (Fig. 6). Replacement of CCG-III) and blocker-type inhibitors [L-TBOA, ETB-TBOA,
Naϩ by Kϩ also resulted in complete loss of the binding, (2S,3S)-3-{3-[4-methoxybenzoylamino]benzyloxy}aspartate
whereas replacement by Liϩ retained approximately half of (PMB-TBOA), and TFB-TBOA] displaced [3H]ETB-TBOA
the specific binding. It is interesting that [14C]glutamate binding. For EAATs1–3, the rank order of inhibition was
uptake by COS-1 cells in LiCl buffer was greatly reduced similar to the uptake assay, with the exception that the Ki
(Ͻ10% compared with NaCl buffer). In the presence of Naϩ, value of glutamate for EAAT3 was unexpectedly high. The
most potent inhibitor for all subtypes was TFB-TBOA. The Ki
values of TFB-TBOA for EAAT1,2,4,5 were in the low nano-
molar range (3.7–14.8 nM), whereas the Ki for EAAT3 was
higher (282 nM). These results were consistent with the
electrophysiological results from the X. laevis oocyte studies
(Shimamoto et al., 2004). Dihydrokainate (DHKA), an
EAAT2-selective blocker, displaced [3H]ETB-TBOA binding
only in EAAT2-expressing cells (Ki ϭ 541 ␮M) and not in the
other subtypes even at concentrations of 1 mM. Although
␣-aminoadipate and CCG-II selectively inhibited glutamate
uptake in the cerebellum (Robinson et al., 1993), these com-
pounds did not affect [3H]ETB-TBOA binding even at con-
centrations of 1 mM.
TABLE 1
Kd and Bmax values of ͓3H͔ETB-TBOA binding to EAAT-expressing
COS-1 cell membranes and rat brain crude membranes
Values were determined from the Scatchard analysis. Data are the mean Ϯ S.E.M.
from n ϭ 3 to 7 experiments.
n
Kd
Bmax
nM
pmol/mg protein
EAAT1
4
7
3
3
4
7
15.5 Ϯ 1.1
16.2 Ϯ 2.5
320 Ϯ 44
11.4 Ϯ 0.7
24.8 Ϯ 5.4
29.5 Ϯ 3.7
14.3 Ϯ 9.7
13.6 Ϯ 3.6
18.9 Ϯ 7.6
2.6 Ϯ 0.5
3.6 Ϯ 0.6
41.1 Ϯ 6.8
EAAT2
EAAT3
EAAT4
EAAT5
Rat brain crude
membranes
Discussion
A binding assay that could evaluate the effects of inhibitors
on all five EAAT subtypes would be very useful for the
evaluation of ligands. [3H]D-aspartate, [3H]L-aspartate, and
[3H](2S,4R)-4-methylglutamate ([3H]4MG) have been used
previously as radioligands for labeling EAAT (Balcar et al.,
1995; Scott et al., 1995; Lieb et al., 2000; Aprico et al., 2001,
2004). However, the Kd values of these ligands were in the
micromolar range and characterization of binding by these
ligands to each subtype has not yet been examined.
In this study, we synthesized [3H]ETB-TBOA as a novel
radioligand. [3H]ETB-TBOA rapidly associated with rat
brain crude membranes and showed significant specific bind-
ing. The radioligand was dissociated by the addition of unla-
beled ligands. The saturation curve fit a single binding-site
model. The Bmax value was approximately 40-fold larger than
Fig. 5. Sodium ion-dependence of [3H]ETB-TBOA binding to EAAT2-
expressing cell membranes. A, saturation plot of [3H]ETB-TBOA binding
in various Naϩ ion concentrations. Specific binding was presented as a
percentage relative to 125 nM [3H]ETB-TBOA binding in 120 mM NaCl
buffer for 30 min. B, specific binding of 20 nM [3H]ETB-TBOA in various
Naϩ concentrations. Specific binding at each concentration was normal-
ized to the amount bound at 120 mM Naϩ (100%). Data represent the
mean Ϯ S.E.M. of at least three independent experiments (n ϭ 3–15).
Fig. 6. Effects of ions on [3H]ETB-TBOA binding to EAAT2-expressing
cell membranes (Ⅺ) and [14C]glutamate uptake by EAAT2-expressing
cells (u). Data represent mean Ϯ S.E.M. of at least three separate exper-
iments.

300
Shimamoto et al.
that of iGluRs in rat brain crude membranes (1.1–1.6 inhibited by (2S,3S)-3-{3-[4-substituted-benzoylamino]ben-
pmol/mg of protein) (London and Coyle, 1979; Murphy et al., zyloxy}aspartate than the other subtypes (Shimamoto et al.,
1987; Sills et al., 1991), which reflects the fact that EAATs 2004). The Bmax values of EAAT4 and EAAT5 were much
are abundant in brain (Danbolt, 2001). The substrate-speci- lower than those of EAATs1–3, which may partly reflect the
ficity in the displacement assays indicates that ETB-TBOA low uptake by EAAT4 and EAAT5.
binds competitively to a substrate binding site. In contrast,
Specific binding by [3H]ETB-TBOA was Naϩ-dependent
GluR agonists did not inhibit [3H]ETB-TBOA binding, and and was completely lost in ChCl buffer. The affinity in-
ETB-TBOA did not affect the activity of iGluRs, mGluRs, or creased with increasing Naϩ-concentrations. These results
vesicular glutamate transporters. Thus, the majority of the indicate that at least one Naϩ ion binds to the transporter
bound radioactivity is attributable to EAATs.
before substrate binding, which is in agreement with a pre-
Significant specific binding was also obtained using indi- vious report (Watzke et al., 2001), and suggest that the Naϩ
vidual EAAT subtypes on EAAT-expressing cell membranes. ion changes the protein conformation to an active form. It is
Each subtype showed the presence of a single binding site. interesting that the Hill coefficient for the Naϩ dependence of
The Kd values for EAAT1,2,4,5 were 10 to 25 nM, whereas [3H]ETB-TBOA binding in this study was approximately 1
that for EAAT3 was more than an order of magnitude higher. (1.10 Ϯ 0.03), indicating that Naϩ binds to a single class of
We have reported previously that EAAT3 was less potently noncooperative binding sites. In contrast, the Hill coefficients
Fig. 7. Displacement of [3H]ETB-TBOA binding by drugs known to interact with EAATs in EAAT-expressing cell membranes and rat brain crude
membranes. Specific binding was normalized to the control (100%). Nonspecific binding was determined in the presence of 10 ␮M TFB-TBOA. Data
represent mean Ϯ S.E.M. of at least three separate experiments. The concentration of [3H]ETB-TBOA used and the Ki values of inhibitors for each
preparation are summarized in Table 2.
TABLE 2
Ki values of various inhibitors of ͓3H͔ETB-TBOA binding
Ki values were determined from the IC50 values determined in Fig. 7. Data are the mean Ϯ S.E.M.
Rat Brain Crude
EAAT1
EAAT2
EAAT3
EAAT4
10 nM
EAAT5
20 nM
Membranes
␮M
͓3H͔ETB-TBOA
20 nM
20 nM
100 nM
10 nM
TFB-TBOA
ETB-TBOA
PMB-TBOA
L-TBOA
L-Glutamate
L-Aspartate
t-2,4-PDC
CCG-II
0.0096 Ϯ 0.0040
0.021 Ϯ 0.0015
0.021 Ϯ 0.0047
26 Ϯ 6.2
0.015 Ϯ 0.0063
0.030 Ϯ 0.0031
0.045 Ϯ 0.0071
6.6 Ϯ 0.9
0.28 Ϯ 0.11
0.71 Ϯ 0.21
1.2 Ϯ 0.30
20 Ϯ 3.5
5065 Ϯ 2830
67 Ϯ 1.4
347 Ϯ 146
Ͼ1000
0.0039 Ϯ 0.0003
0.0068 Ϯ 0.0009
0.026 Ϯ 0.0042
3.9 Ϯ 0.59
26 Ϯ 2.5
0.0037 Ϯ 0.0004
0.025 Ϯ 0.0039
0.095 Ϯ 0.016
3.9 Ϯ 0.61
420 Ϯ 80
0.0027 Ϯ 0.0004
0.015 Ϯ 0.0016
0.020 Ϯ 0.0034
8.4 Ϯ 1.3
259 Ϯ 49
137 Ϯ 17
490 Ϯ 44
47 Ϯ 10
125 Ϯ 8.7
50 Ϯ 13
Ͼ1000
10 Ϯ 2.2
541 Ϯ 3.0
Ͼ1000
8.5 Ϯ 1.2
44 Ϯ 3.7
44 Ϯ 4.7
144 Ϯ 44
23 Ϯ 2.5
Ͼ1000
2.8 Ϯ 0.16
Ͼ1000
Ͼ1000
403 Ϯ 82
26 Ϯ 4.0
Ͼ1000
Ͼ1000
Ͼ1000
CCG-III
DHKA
␣-Aminoadipate
9.2 Ϯ 0.8
21 Ϯ 1.0
Ͼ1000
Ͼ1000
11 Ϯ 2.5
4.8 Ϯ 0.7
Ͼ1000
Ͼ1000
Ͼ1000
3324 Ϯ 442
Ͼ1000
Ͼ1000
t-2,4-PDC, L-trans-pyrrolidine-2,4-dicarboxylic acid.

Glutamate Transporter Binding Assay
301
for substrate transport (L-glutamate or D-aspartate), mea- was, however, weaker than inhibition of binding to EAAT2-
sured by uptake assays or electrophysiological studies, were expressing cell membranes. EAAT2 is responsible for 30 to
reported to be 1.9 to 3.3, which most likely reflects the re- 50% of [3H]4MG binding in rat brain membranes (Aprico et
quirement for two to three Naϩ ions to complete the uptake al., 2001). Recently EAAT2-preferring blockers have been
process (Klo¨ckner et al., 1993; Sugawara et al., 1998; Kim- reported (Dunlop et al., 2003, 2005; Bunch et al., 2006), but
mich et al., 2001). These results suggest that Naϩ binding in their effects on EAAT4 and EAAT5 are unknown. Character-
the ligand binding step is different from Naϩ binding in the ization of these compounds in the binding assay developed
substrate-uptake process. ETB-TBOA is a blocker and is here may further elucidate their specificity. Although
involved only in the ligand binding step. Additional Naϩ ion (2S,1ЈR,2ЈR)-2-(2-carboxycyclopropyl)glycine (CCG-II) and
binding steps would be necessary for subsequent steps in the ␣-aminoadipate were reported to inhibit glutamate uptake in
transport process. Moreover, similar to previous observa- the cerebellum (Robinson et al., 1993) and ␣-aminoadipate
tions, Liϩ ions could partly substitute for Naϩ ions (Borre was used as a substrate for neuronal transporters in cerebel-
and Kanner, 2001; Larsson et al., 2004). In contrast, Kϩ lum preparations, they did not bind to EAAT4 even at con-
could not be replaced by Naϩ, probably because of its larger centrations of 1 mM. Because ␣-aminoadipate also inhibits
ion radius. Kϩ was not essential for substrate binding, al- the glutamate-cystine antiporter, its effects on EAATs need
though it is indispensable for uptake. In addition, the ab- to be verified in electrophysiological studies.
sence of ClϪ ion did not affect binding.
The most potent EAAT inhibitor tested here was TFB-
EAAT inhibitors displaced [3H]ETB-TBOA in EAAT-ex- TBOA (Shimamoto et al., 2004). The Ki values of TFB-TBOA
pressing cell membranes and in rat brain crude membranes. for [3H]ETB-TBOA binding to EAATs1–3 (9.6, 15, and 282
The Ki values were very similar to the IC50 values reported nM, respectively) were similar in magnitude to the IC50
previously in uptake assays using EAATs1–3 (Arriza et al., values determined by the glutamate uptake assay (22, 17,
1994; Shimamoto et al., 1998), whereas some values were and 300 nM, respectively). TFB-TBOA also showed a high
higher than those obtained from [3H]4MG or [3H]D-aspartate affinity for EAAT4 and EAAT5 (3.9 and 3.7 nM, respectively).
binding in rat brain membrane homogenates or astrocytic As expected from the results of the uptake assays, ETB-
membrane homogenates (Aprico et al., 2001, 2004). This dis- TBOA and PMB-TBOA were slightly less potent in binding to
crepancy may be due to the fact that the concentrations of EAATs1–3 than TFB-TBOA. It is interesting that the affinity
[3H]4MG or [3H]D-aspartate (40 nM) in the previous studies of PMB-TBOA for EAAT5 was markedly lower than that of
were much lower than the Kd values of the compounds (6.0 TFB-TBOA. The binding affinity for EAAT5 was strongly
and 15.0 ␮M, respectively), whereas, in the present experi- affected by substituents. Recently, Takayasu et al. (2006)
ments, the concentrations (10–100 nM) of [3H]ETB-TBOA reported that PMB-TBOA blocked the transport current
were similar to the Kd values. The overall rank order of evoked by stimulation of the climbing fiber in Bergmann glia
activity was similar to previous uptake assays and electro- approximately 10-fold more potently than in Purkinje cells.
physiological studies, with the exception that the affinity of EAAT4 is preferentially involved in clearing glutamate from
L-glutamate for EAAT3 was unexpectedly low. The Ki value climbing fiber-Purkinje cell synapses (Huang et al., 2004;
of L-glutamate for EAAT3 was more than 5 mM, which is Takayasu et al., 2005). In the present study, however, the
approximately 180 times larger than the Km value obtained affinity of PMB-TBOA for EAAT4 was comparable with its
from the electrophysiological assay (28 ␮M), whereas L-as- affinity for the glial transporters (EAAT1 and EAAT2), sug-
partate showed a Ki value (67 ␮M) that would be reasonably gesting that unknown mechanisms regulate the affinity in
expected from its Km value (24 ␮M) (Arriza et al., 1994). vivo. Development of EAAT4-selective inhibitors would elu-
Because the affinity of [3H]ETB-TBOA for EAAT3 is low, cidate these discrepancies.
higher-affinity radiolabeled ligands for EAAT3 would be nec-
In conclusion, the [3H]ETB-TBOA binding assay will pro-
essary to further elucidate the molecular basis for the dis- vide useful pharmacological information about EAATs. The
crepancies. Although the differences were not as great as binding assay using frozen brain crude membranes is much
that seen with EAAT3, L-aspartate showed higher affinity easier to conduct than the uptake assay using freshly
than L-glutamate in all subtypes.
prepared synaptosomes, and thus, it is applicable to high-
The binding site of EAATs recognizes the folded form of throughput screening to find novel EAAT ligands or to ex-
L-glutamate or the anti-form of aspartates (Bridges and Es- amine the side effects of drugs on EAATs. [3H]D-aspartate
slinger, 2005), and many analogs mimicking these conforma- has been used for comparison of synaptosomal preparations
tions have been designed. One of the folded-type glutamate between nondemented control and diseased brain samples
analogs, L-CCG-III (Nakamura et al., 1993), inhibited (Scott et al., 1995), and [3H]L-aspartate has been used for
[3H]ETB-TBOA binding to all subtypes with moderate po- autoradiographic studies (Lieb et al., 2000). Because the
tency (low micromolar concentration). The other isomers, binding affinity of [3H]ETB-TBOA is much higher than bind-
L-CCG-I, II, and IV, did not markedly inhibit [3H]ETB-TBOA ing affinities of the aspartates and [3H]ETB-TBOA does not
binding even at concentrations of 1 mM. Evaluation of sub- interact with other proteins, similar applications are possi-
type selective inhibitors is one of the important roles of the ble. In addition, the lack of a simple assay system to evaluate
assay. DHKA, a well-known EAAT2-selective blocker, dis- the compounds binding to EAAT4 and EAAT5 has prevented
placed [3H]ETB-TBOA binding to EAAT2 only at a concen- the full characterization of subtype-selective inhibitors. Us-
tration of 1 mM, although it was less potent in our prepara- ing cell membranes expressing each EAAT subtype, this
tions than in other reports (Shimamoto et al., 1998). binding assay has enabled direct and quantitative compari-
Glutamate uptake into synaptosomes is inhibited by DHKA, sons among all subtypes. Thus, [3H]ETB-TBOA should serve
suggesting EAAT2 is primarily responsible. The inhibition by as a useful tool to test newly developed subtype-selective
DHKA of [3H]ETB-TBOA binding in rat crude membranes EAAT ligands and will result in a better understanding of

302
Shimamoto et al.
Kimmich GA, Roussie J, Manglapus M, and Randles J (2001) Characterization of
Naϩ-coupled glutamate/aspartate transport by a rat brain astrocytes line express-
ing GLAST and EAAC1. J Membr Biol 182:17–30.
synaptic transmission mechanisms and the etiology of neu-
ronal disorders.
Klo¨ckner U, Storck T, Conradt M, and Stoffel W (1993) Electrogenic L-glutamate
uptake in Xenopus laevis oocytes expressing a cloned rat brain L-glutamate/L-
aspartate transporter (GLAST-1). J Biol Chem 268:14594–14596.
Larsson HP, Tzingounis AV, Koch HP, and Kavanaugh MP (2004) Fluorometric
measurements of conformational changes in glutamate transporters. Proc Natl
Acad Sci USA 101:3951–3956.
Levy LM, Warr O, and Attwell D (1998) Stoichiometry of the glial glutamate
transporter GLT-1 expressed inducibly in a Chinese hamster ovary cell line se-
lected for low endogenous Naϩ-dependent glutamate uptake. J Neurosci 18:9620–
9628.
Lieb I, Chebib M, Cooper B, Dias LS, and Balcar VJ (2000) Quantitative autoradiog-
raphy of Naϩ-dependent [3H]L-aspartate binding to L-glutamate transporters in
rat brain: structure-activity studies using L-trans-pyrrolidine-2,4-dicarboxylate
(L-t-PDC) and 2-(carboxycyclopropyl)-glycine (CCG). Neurochem Int 36:319–327.
London ED and Coyle JT (1979) Specific binding of [3H]kainic acid to receptor sites
in rat brain. Mol Pharmacol 15:492–505.
Acknowledgments
We thank Professor E. Gouaux (Oregon Health and Science Uni-
versity, Portland, OR) for providing useful information about sodium
coupling. We thank Professor T. Ueda and Dr. D. Bole (University of
Michigan, Ann Arbor, MI) for conducting the vesicular glutamate
uptake assay. We also thank Professor S. G. Amara (University of
Pittsburgh, Pittsburgh, PA) for providing MDCK cells expressing
EAAT2 or EAAT3 and cDNA of pOTV-EAAT5, Professor S. Nakan-
ishi (Osaka Bioscience Institute, Osaka, Japan) for providing Chi-
nese hamster ovary cells expressing mGluRs, and T. Maeda (Suntory
Institute for Bioorganic Research, Osaka, Japan) for technical assis-
tance.
Murphy DE, Snowhill EW, and Williams M (1987) Characterization of quisqualate
recognition sites in rat brain tissue using DL-[3H]alpha-amino-3-hydroxy-5-
methylisoxazole-4-propionic acid (AMPA) and a filtration assay. Neurochem Res
12:775–782.
References
Aprico K, Beart PM, Lawrence AJ, Crawford D, and O’Shea RD (2001) [3H](2S,4R)-
4-Methylglutamate: a novel ligand for the characterization of glutamate transport-
ers. J Neurochem 77:1218–1225.
Nakamura Y, Kataoka K, Ishida M, and Shinozaki H (1993) (2S,3S,4R)-2-
(carboxycyclopropyl)glycine, a potent and competitive inhibitor of both glial and
neuronal uptake of glutamate. Neuropharmacology 32:833–837.
Robinson MB, Sinor JD, Dowd LA, and Kerwin JF Jr (1993) Subtypes of sodium-
dependent high-affinity L-[3H]glutamate transport activity: pharmacologic speci-
ficity and regulation by sodium and potassium. J Neurochem 60:167–179.
Sakai R, Swanson GT, Shimamoto K, Green T, Contractor A, Ghetti A, Tamura-
Horikawa Y, Oiwa C, and Kamiya H (2001) 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 296:
650–658.
Scott HL, Tannenberg AEG, and Dodd PR (1995) Variant forms of neuronal gluta-
mate transporter sites in Alzheimer’s disease cerebral cortex. J Neurochem 64:
2193–2202.
Shigeri Y, Seal RP, and Shimamoto K (2004) Molecular pharmacology of glutamate
transporters, EAATs and VGLUTs. Brain Res Rev 45:250–265.
Shigeri Y, Shimamoto K, Yasuda-Kamatani Y, Seal RP, Yumoto N, Nakajima T, and
Amara SG (2001) Effects of threo-␤-hydroxyaspartate derivatives on excitatory
amino acid transporters (EAAT4 and EAAT5). J Neurochem 79:297–302.
Shimamoto K, Lebrun B, Yasuda-Kamatani Y, Sakaitani M, Shigeri Y, Yumoto N,
and Nakajima T (1998) DL-threo-␤-Benzyloxyaspartate, a potent blocker of excita-
tory amino acid transporters. Mol Pharmacol 53:195–201.
Shimamoto K, Sakai R, Takaoka K, Yumoto N, Nakajima T, Amara SG, and Shigeri
Y (2004) Characterization of novel L-threo-␤-benzyloxyaspartate derivatives, po-
tent blockers of the glutamate transporters. Mol Pharmacol 65:1008–1015.
Shimamoto K and Shigeri Y (2006) Elucidation of the glutamate transporter func-
tions using selective inhibitors. CNS Agents Med Chem 6:59–71.
Sills MA, Fagg G, Pozza M, Angst C, Brundish DE, Hurt SD, Wilusz EJ, and
Williams M (1991) [3H]CGP 39653: a new N-methyl-D-aspartate antagonist radio-
ligand with low nanomolar affinity in rat brain. Eur J Pharmacol 192:19–24.
Sugawara M, Kato M, Kobayashi M, Iseki K, and Miyazaki K (1998) Mechanism of
the inhibitory effect of imipramine on the Naϩ-dependent transport of L-glutamic
acid in rat intestinal brush-border membrane. Biochim Biophys Acta 1370:252–
258.
Takayasu Y, Iino M, Kakegawa W, Maeno H, WataseK, Wada K, Yanagihara D,
Miyazaki T, Momine O, Watanabe M, et al. (2005) Differential roles of glial and
neuronal glutamate transporters in Purkinje cell synapses. J Neurosci 25:8788–
8793.
Takayasu Y, Iino M, Shimamoto K, Tanaka K, and Ozawa S (2006) Glial glutamate
transporters maintain one-to-one relationship at the climbing fiber-Purkinje cell
synapse by preventing glutamate spillover. J Neurosci 26:6563–6572.
Wadiche JI, Amara SG, and Kavanaugh MP (1995) Ion fluxes associated with
excitatory amino acid transport. Neuron 5:721–728.
Aprico K, Beart PM, Crawford D, and O’Shea RD (2004) Binding and transport of
[3H](2S,4R)-4-methylglutamate, a new ligand for glutamate transporters, demon-
strate labeling of EAAT1 in cultured murine astrocytes. J Neurosci Res 75:751–
759.
Arriza JL, Eliasof S, Kavanaugh MP, and Amara SG (1997) Excitatory amino acid
transporter 5, a retinal glutamate transporter coupled to a chloride conductance.
Proc Natl Acad Sci USA 94:4155–4160.
Arriza JL, Fairman WA, Wadiche JI, Murdoch GH, Kavanaugh MP, and Amara SG
(1994) Functional comparisons of three glutamate transporter subtypes cloned
from human motor cortex. J Neurosci 14:5559–5569.
Balcar VJ, Li Y, and Killinger
S (1995) Effects of L-trans-pyrrolidine-2,4-
dicarboxylate and L-threo-3-hydroxyaspartate on the binding of [3H]L-aspartate,
[3H]alpha-amino-3-hydroxy-5-methyl-4-isoxazole- propionate (AMPA), [3H]DL-E,-
2-amino-4-propyl-5-phosphono-3-pentenoate (CGP 39653), [3H]6- cyano-7-
nitroquinoxaline-2,3-dione (CNQX) and [3H]kainate studied by autoradiography
in rat forebrain. Neurochem Int 26:155–164.
Bergles DE, Tzingounis AV, and Jahr CE (2002) Comparison of coupled and uncou-
pled currents during glutamate uptake by GLT-1 transporters. J Neurosci 22:
10153–10162.
Borre L and Kanner BI (2001) Coupled, but not uncoupled, fluxes in a neuronal
glutamate transporter can be activated by lithium ions. J Biol Chem 276:40396–
40401.
Bridges RJ and Esslinger CS (2005) The excitatory amino acid transporters: phar-
macological insights on substrate and inhibitor specificity of the EAAT subtypes.
Pharmacol Ther 107:271–285.
Bunch L, Nielsen B, Jensen AA, and Brauner-Osborne H (2006) Rational design and
enantioselective synthesis of (1R,4S,5R,6S)-3-azabicyclo[3.3.0]octane-4,6-
dicarboxylic acid—a novel inhibitor at human glutamate transporter subtypes 1, 2,
and 3. J Med Chem 49:172–178.
Campiani G, Fattorusso C, De Angelis M, Catalanotti B, Butini S, Fattorusso R,
Fiorini I, Nacci V, and Novellino
dependent glutamate transporters (EAATs): targets for the development of novel
therapeutics against neurodegenerative diseases. Curr Pharm Des 9:599–625.
Danbolt NC (2001) Glutamate uptake. Prog Neurobiol 65:1–105.
Dunlop J, Eliasof S, Stack G, McIlvain HB, Greenfield A, Kowal D, Petroski R, and
Carrick T (2003) WAY-855 (3-amino-tricyclo[2.2.1.02.6]heptane-1,3-dicarboxylic
acid): a novel, EAAT2-preferring, nonsubstrate inhibitor of high-affinity glutamate
uptake. Br J Pharmacol 140:839–846.
Dunlop J, McIlvain HB, Carrick TA, Jow B, Lu Q, Kowal D, Lin S, Greenfield A,
Grosanu C, Fan K, et al. (2005) 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 68:974–982.
Eliasof S, McIlvan HB, Petroski RE, Foster AC, and Dunlop J (2001) Pharmacolog-
ical characterization of threo-3-methylglutamic acid with excitatory amino acid
transporters in native and recombinant systems. J Neurochem 77:550–557.
Fairman WA, Vandenberg RJ, Arriza JL, Kavanaugh MP, and Amara SG (1995) An
excitatory amino-acid transporter with properties of a ligand-gated chloride chan-
nel. Nature (Lond) 375:599–603.
E (2003) Neuronal high-affinity sodium-
Watzke N, Bamberg E, and Grewer C (2001) Early intermediates in the transport
cycle of the neuronal excitatory amino acid carrier EAAC1. J Gen Physiol 117:
547–562.
Zerangue N and Kavanaugh MP (1996) Flux coupling in a neuronal glutamate
transporter. Nature (Lond) 383:634–637.
Address correspondence to: Dr. Keiko Shimamoto, Suntory Institute for
Bioorganic Research, 1-1-1, Wakayamadai, Shimamoto-cho, Mishima-gun,
Osaka 618-8503. E-mail: shimamot@sunbor.or.jp
Huang YH, Dykes-Hoberg M, Tanaka K, Rothstein JD, and Bergles DE (2004)
Climbing fiber activation of EAAT4 transporters and kainate receptors in cerebel-
lar Purkinje cells. J Neurosci 24:103–111.