(Received for publication, May 18, 1995; and in revised form, July 17, 1995)
From the
The reuptake of excitatory amino acids, such as glutamate,
terminates excitatory signals and prevents the persistence of
excitotoxic levels of glutamate in the synaptic cleft. The L-glutamate/L-aspartate transporter (GLAST-1) is the
first member of the recently discovered glutamate transporter family,
which includes GLT-1 and EAAC1. The neutral amino acid carrier ASCT1 is
structurally closely related to this new family of membrane proteins.
Transmembrane transport of neutral amino acids is expected to differ in
its binding site from that of the acidic excitatory amino acids
glutamate and aspartate. Three positively charged amino acid residues,
Arg-122, Arg-280, Arg-479, and one polar Tyr-405 are conserved in all
glutamate transporters. They are replaced by apolar amino acid residues
in the ASCT1 sequence. We exchanged these residues in the
GLAST-1-specific cDNA by site-directed mutagenesis. cRNAs of these
mutants were expressed in the Xenopus oocyte system. The
functional characterization of the mutants R122I and R280V and the
double mutant R122I,R280V revealed that the mutations have no influence
on the intrinsic properties and kinetics of glutamate transport but
alter the K-values for L-aspartate and the competitive inhibitor D,L-threo-3-hydroxy aspartate. Substitutions of
Tyr-405 by Phe (Y405F) and Arg-479 (R479T) by Thr completely inactivate
the glutamate transporter. Immunoprecipitations of
[
S]methionine-labeled transporter molecules
indicate similar expression levels of wild-type and mutant
transporters. Immunostaining of oocyte sections clearly proves the
correct targeting to and integration of the mutant GLAST-1 proteins in
the plasma membrane. Our results suggest the pivotal function of the
hydroxy group of the highly conserved Tyr-405 and the positively
charged Arg-479 in the binding of the negatively charged acidic
neurotransmitter glutamate.
The regulation of the neurotransmitter concentration in the
synaptic cleft is an important component of the synaptic transmission
process(1, 2) . It is mediated by high affinity,
Na-dependent uptake systems. The neurotransmitter
transporters have been characterized by the uptake of radiolabeled
substrates in brain slices(1) , synaptosomes(3) , and
isolated cells (4) for their substrate specificity and ion
dependence. A number of neurotransmitter carriers have been cloned on
the basis of structural homologies and found to form a family of
related proteins (5, 6) . The recently discovered
Na
-dependent glutamate transporters (GLAST-1, GLT-1,
and EAAC1) represent a new family of integral membrane proteins (7, 8, 9) with approximately 50% amino acid
identities. They show significant similarities ranging between 27 and
32% to the proton L-glutamate transporter protein (GLTP) of Escherichia coli(10) , Bacillus
stearothermophilus, and Bacillus caldotenax and to the
dicarboxylate transporter (DCTA) of Rhizobium
meliloti(11) .
Models proposed for the membrane integration of the glutamate transporters show a consensus regarding six N-terminal transmembrane helices with a large extracellular loop between the proposed transmembrane helices 3 and 4. We have shown that two of three putative N-glycosylation sites of GLAST-1 localized in this extracellular loop are N-glycosylated. These sites are also common to GLT-1, EAAC1, and ASCT1, and it is reasonable to suggest that they are glycosylated in a similar manner(12) . On the other hand, the topology of the highly conserved C-terminal part of the protein awaits experimental clarification(13) .
The cloned transporter GLAST-1 cotransports glutamate with three sodium ions across the plasma membrane, whereas one potassium ion is countertransported out of the cell(14) . There is evidence from studies in the salamander retinal glia that a pH-changing anion, probably a hydroxyl ion, is countertransported(15) . Despite the extensive electrophysiological characterization of the transport process (16, 17, 18) , it remains unclear which amino acid residues of GLAST-1 are involved in the uptake process. It is reasonable to assume that charged or polar residues of the glutamate transporter are involved in the binding and translocation of glutamate and its cosubstrates. Mutagenesis has been carried out on Lys-298 and His-326 of the glutamate transporter GLT1. Substitution of these two residues with polar and positively charged residues leads (in the case of Lys-298) to a reduced transport activity, which has been interpreted as a targeting defect, whereas the severely impaired transport by GLT-1 mutated at His-326 has been referred to a participation of His-326 in the putative proton translocation process of the transporter(19) .
Neutral amino acids in mammalian
cells are predominantly transported by two
Na-dependent uptake systems ASC (predominantly Ala,
Ser, and Cys) and A (predominantly Ala). The recently cloned neutral
amino acid transporter ASCT1 (20) also called SATT (21) resembles the properties of the ASC transporter and
displays
37% sequence identity with the structurally related
excitatory amino acid transporter family.
In the present study, we
analyzed the contribution of positively charged or polar residues of
GLAST-1 to the binding and/or transport of the negatively charged
neurotransmitter glutamate. We considered charged or polar residues of
GLAST-1, which are conserved throughout the eukaryotic and prokaryotic
glutamate transporters but are replaced in the neutral amino acid
transporter by apolar residues. These residues were substituted by the
amino acid residues present at the respective sites of the sequence of
ASCT1. We determined the K values for
glutamate and Na
and measured the voltage dependence
of these mutant GLAST-1 in the Xenopus oocyte system.
Mutagenized GLAST-1 with the substitutions R122I and R280V and the
double mutant R122I,R280V exhibits nearly unaffected transport kinetics
as compared to the wild type. However the K
values for THA (
)and aspartate of all three
mutants are changed in a similar manner. GLAST-1 mutants Y405F and
R479T and the quadruple mutant R122I,R280V,Y405F,R479T (Q) revealed
that Tyr-405 and Arg-479 are essential for glutamate transport
activity. We propose that the hydroxy group of the conserved Tyr-405
and the positively charged Arg-479 contribute to the binding of the
acidic neurotransmitter glutamate.
Figure 1:
Alignment of the amino acid sequences
of mammalian glutamate transporters GLAST-1, EAAC1, GLT-1, neutral
amino acid transporter ASCT1, and prokaryotic DCTA dicarboxylate
transporter of R. meliloti. The amino acid sequences are
deduced from the cDNA nucleotide sequences. Sequence
identities/similarities are indicated by using white on black
lettering/black on gray lettering, respectively. The
experimentally verified N-glycosylation sites of GLAST-1 are
marked by blacktriangles. Lines are drawn
over the N-terminal six hydrophobic regions, which are proposed to span
the plasma membrane in a -helical manner. The amino acid
substitutions are indicated by blackcircles.
Sequences of GLAST-1, EAAC1, GLT-1, ASCT1, and DCTA are taken from the
EMBL/GenBank data base under accession numbers X6374, L12411, X67857,
L14595, and P20672 respectively.
Figure 2:
Immunoprecipitation of wild-type and
mutant GLAST-1 proteins expressed in Xenopus oocytes.
Wild-type and mutant GLAST-1 cRNAs were injected into Xenopus oocytes and incubated in Barth's medium supplemented with
[S]methionine. Oocytes were homogenized and
immunoprecipitated as described under ``Experimental
Procedures.'' Samples were analyzed by SDS-polyacrylamide gel
electrophoresis and fluorography. Sizes of marker proteins are
indicated in kDa. Lane1, water-injected control
oocytes; lanes2-6, indicated mutants; lane7, double mutant R122I,R280V (D); lane8, quadruple mutant Q.
Figure 3:
Comparison of the basic L-glutamate transport properties of wild-type and mutant
GLAST-1. A, L-Glu K values. The values represent the means, and the bars represent the standard deviation of I
(n = 4-11). The current of each oocyte was
normalized to the current amplitude at 100 µML-Glu. The solidline is fitted to the
data of wild-type GLAST-1 by minimizing squared errors according to the
equation I = I
[S]
/([S]
+ K
) with an apparent K
value of 21 ± 3 µML-glutamate. The best fit to the data of the mutant
transporters yielded nearly identical concentration response curves (K
values for L-glutamate:
R122I, 22.5 ± 2.5 µM; R280V, 18 ± 4
µM; R122I,R280V, 18 ± 3 µM). The
cooperativity coefficients were between 1 and 1.3 ± 0.1. The
holding potential was -90 mV (90 mM [Na
]
). B,
concentration response curve for Na
. The values
represent the means, and the bars represent the standard
deviation from 4-6 oocytes. The current of each oocyte was
normalized to the current amplitude at 90 mM
[Na
]
. The solidline was fitted to the data of wild-type GLAST-1 by
minimizing squared errors according to the equation mentioned under
``Experimental Procedures'' with an apparent K
value of 32 ± 3 mM (cooperativity coefficient = 1.8). The best fit to the data
obtained from the mutants R122I, R280V, and R122I,R280V yielded a
nearly identical concentration response curve with K values
of 36 ± 5, 31 ± 1, and 29 ± 5 mM,
respectively (cooperativity coefficient = 1.8 ± 0.3). The
currents were recorded at 100 µML-Glu (holding
potential, -90 mV). Only the data of the double mutant were
plotted. C, voltage dependence of I
. Data
shown are mean ± S.D. obtained from 4-6 different oocytes.
Currents were normalized to the current amplitude at -90 mV. The solidline was fitted by eye.
[L-Glu] and [Na
] were 100
µM and 90 mM,
respectively.
Figure 5:
Functional characterization of the
mutant transporters Y405F and R479T. A, original traces
recorded at indicated L-Glu concentrations. Superfusion of
oocytes expressing Y405F, R479T, and Q with 500 µML-Glu evoked no detectable currents in contrast to
wild-type GLAST-1-expressing oocytes. [L-Glu] up to
500 mM revealed the same results (data not shown). B, L-[C]glutamate uptake mediated by
wild-type GLAST-1 and mutant transporters. The L-[
C]glutamate uptake rates of R122I
and R280V and the double mutant R122I,R280V are quite similar as
compared to the L-[
C]glutamate uptake
of wild-type GLAST-1. Y405F, R479T, and the quadruple mutant
Q-expressing oocytes show L-[
C]glutamate uptake rates similar to
water-injected controls. C, L-[
C]alanine uptake by wild-type
GLAST-1 and mutant transporters is similar to the water-injected
control oocytes. Each column represents the mean ± S.D. (n = 4).
We studied the Na dependence of the mutant transporters to probe for electrostatic
forces between the sodium ion and the two positively charged arginines.
Inward currents were recorded at stepwise increased
[Na
]
and constant L-Glu
concentration (100 µM). Currents were fitted to a Hill
equation, which yielded K
values for
Na
of wild type and the double mutant R122I,R280V of 32
± 3 mM (n = 6) and 29 ± 5 mM (n = 4), respectively (Fig. 3B).
The values of the mutant transporter R122I and R280V for Na
are neither significantly affected (R122I, 36 ± 5
mM, n = 4; R280V, 31 ± 1 mM, n = 2).
Glutamate transport is thought to be associated with conformational changes within the electrical transmembrane field. The influence of positively charged side chains as compared to neutral amino acid residues on the voltage dependence of the transport process has been explored in the experiment depicted in Fig. 3C. The peak currents of wild type and the double mutant R122I,R280V were plotted as a function of voltage. The resulting curves are roughly superimposed. This suggests that the elimination of two positive charges at amino acid positions 122 and 280 of GLAST-1 has no detectable effect on the voltage dependence of the transport process. The results for the mutants R122I and R280V were quite similar (data not shown).
However, the expressed mutant GLAST-1 transporters
R122I and R280V as well as the resulting double mutant show a
significant decrease (paired t test, p < 0.05) in
the apparent K value for the competitive inhibitor
THA from 22 ± 6 µM (n = 6) for
wild-type GLAST-1 to 10 ± 2 µM (n =
6), 11 ± 3 µM (n = 6), 10 ±
2 µM (n = 6) for R122I, R280V and
R122I,R280V, respectively (Fig. 4A). The increase of
the apparent affinity for L-aspartate is similar as to the
value for THA (Fig. 4B). The K
value for L-aspartate of wild-type GLAST-1 is 14.5
± 2 µM (n = 4) and 9.5 ± 0.9
µM (n = 4) (significant smaller paired t test, p < 0.05) for the double mutant
R122I,R280V. L-[
C]Alanine uptake
experiments shown in Fig. 5C and electrophysiological
measurements (data not shown) revealed that substitution of Arg-122 and
280 by Ile and Val, respectively does not enable GLAST-1 to transport
neutral amino acids (Ala, Ser, Cys, and Thr) with a higher efficiency
than the controls.
Figure 4:
K values of
THA (A) and L-aspartate (B) for wild-type
and mutant GLAST-1 transporters. Dose response curves as described in Fig. 3A, except that THA and L-Asp were
applicated instead of L-glutamate. The K
values for THA and L-Asp are significantly smaller
for the mutants R122I and R280V and the double mutant R280V,R122I
(paired t test; p = 0.03 and p = 0.01, respectively). The K
value for THA of wild-type GLAST-1 is 22 ± 6
µM (n = 6); for the mutants R122I, R280V,
and R122I,R280V, K
values are 10 ±
2 µM (n = 6), 11 ± 3 µM (n = 6), and 10 ± 2 µM (n = 6), respectively. The apparent K
value for aspartate of wild-type GLAST-1 is 14.5 ± 2
µM (n = 4), and the value determined for
the double mutant R122I,R280V is 9.5 ± 0.9 µM (n = 4). The cooperativity coefficients were
between 1 and 1.3 ± 0.1. The holding potential was -90 mV
(90 mM [Na
]
).
Figure 6: Wild-type and mutant GLAST-1 are located in the plasma membrane. Cryosections (10-12 µm) of Xenopus oocytes were incubated with anti-GLAST-1 antibody and stained with fluorescein isothiocyanate-conjugated secondary antibody. Surface expression was noted in wild-type GLAST-1 (B), Y405F (C), R479T (D), and Q (E) expressing oocytes with similar intensity (marked by arrow). In the case of water-injected control oocytes (A), no fluorescence staining was observed (marked by arrow).
The recently discovered three L-glutamate
transporters of central nervous system (7, 8, 9) and the neutral amino acid
transport protein ASCT1 (20, 21) form a family of
integral membrane proteins. They exhibit significant similarity to the
prokaryotic proton L-glutamate transporter protein (GLTP) (10) and to dicarboxylate transporter (DCTA)(11) . The
hydropathy plots of the related eukaryotic transporters suggest a
conserved membrane topology implicating a similar transport
mechanism(26) . The striking difference between the substrates
of the neutral (e.g. alanine) and the acidic amino acid
transporters (e.g. glutamate and aspartate) is the negatively
charged carboxy group of the acidic neurotransmitter. Intriguing amino
acid residues important for the recognition and discrimination of the
different substrates are positively charged or polar. The amino acid
residues Arg-122, Arg-280, Tyr-405, and Arg-479 are conserved
throughout the eukaryotic and prokaryotic glutamate transporters. They
are substituted by apolar residues in the neutral amino acid
transporter ASCT1 (Fig. 1). In the study described here, we
exchanged these amino acid residues of GLAST-1 for the residues of the
neutral amino acid transporter ASCT1. The mutants R122I and R280V as
well as the double mutant R122I,R280V expressed in the Xenopus oocyte system show no significant differences in their apparent
affinity for L-Glu and their Na and voltage
dependence. GLAST-1 mutants R122I and R280V and the double mutant do
not transport any substrate of the neutral amino acid carrier. This
argues against a contribution of Arg-122 and Arg-280 to the substrate
specificity of GLAST-1. Interestingly, the apparent K
values for aspartate and the competitive inhibitor THA are
decreased significantly. Arg-122 and Arg-280 are positioned at the
boundaries between the putative intracellular hydrophilic loops and
transmembrane domains 3 and 5, respectively. Although Arg-122 and -280
seem to be localized intracellularly, we included these residues in our
investigations because transport is possibly mediated by conformational
changes sequentially exposing the substrate binding site to the
external and internal surfaces of the
protein(27, 28) . Charged residues frequently border
hydrophobic regions of integral membrane proteins and thus contribute
to their correct positioning within the membrane(29) . A
conformational change evoked by the lack of the charged residues in the
mutant transporters might facilitate the transport of the less bulky
substrates THA and aspartate in contrast to glutamate.
In contrast to Arg-122 and -280, which are localized in the N-terminal part of GLAST-1, Tyr-405 and Arg-479 are positioned in the C-terminal part of the protein. The topology of the C terminus derived from hydropathy plots is undefined(13, 26) . Therefore, predictions concerning the extra- or intracellular localization of Tyr-405 or Arg-479 are not possible. The C-terminal domain between residues 360-497 is highly conserved, which might indicate a functional importance of this region. This is supported by our data. Substitution of the Tyr-405 and the Arg-479 by Phe and Thr, which are the respective amino acid residues of the neutral amino acid transporter ASCT1, completely abolished L-glutamate transport. Since the expression level of the mutant transporter and the targeting to the plasma membrane is unimpaired ( Fig. 2and Fig. 6), our results strongly suggest that Tyr-405 and Arg-479 are essential for the glutamate transport process. A drawback to mutagenesis studies might be conformational changes of the protein introduced by amino acid substitution, which could complicate the interpretation of the direct role of the amino acid residue in the binding and transport of glutamate. The exchange of Tyr for Phe is nearly conservative. In addition, Phe (Y405F) as well as Thr (R479T) represent amino acids that are localized at identical positions in a highly conserved region of the related neutral amino acid transporter exhibiting a similar hydropathy plot. These facts argue against conformational changes of the affected protein domains.
Our interpretation is supported by
modeling studies on the Clostridium symbiosum glutamate
dehydrogenase structure. They implicate interactions of the
-carboxylate group of glutamate with the
-OH of Ser-380 and
Thr-193 and the
-NH
of
Lys-89(30) . This is in line with our results that the hydroxy
group of Tyr-405 and the positively charged Arg-479 are essential for
glutamate transport. It is conceivable that these amino acid residues,
strongly conserved in the glutamate transporters but not in the neutral
amino acid transporter ASCT1, interact with the
-carboxylate group
of glutamate.
The studies presented here provide new insights into the structure-function relationship of the glutamate transporter family. Further experiments will unravel the structural motifs involved in binding, substrate specificity, and translocation of the L-glutamate transporter.