Glutamate transporters remove this transmitter
from the extracellular space by cotransport with three sodium ions and
a proton. The cycle is completed by translocation of a potassium ion in the opposite direction. Recently we have identified two adjacent amino
acid residues of the glutamate transporter GLT-1 that influence potassium coupling. Using the scanning cysteine accessibility method we
have now explored the highly conserved region surrounding them.
Replacement of each of the five consecutive residues 396-400 by
cysteine abolished transport activity but at several other positions
the substitution is tolerated. One residue, tyrosine 403, was
identified where cysteine substitution renders the transporter sensitive to modification by positively charged methanethiosulfonate derivates in a sodium-protectable fashion. In the presence of sodium,
the nontransported glutamate analogue dihydrokainate potentiated the
covalent modification, presumably by binding to the glutamate site and
locking the protein in a conformation in which tyrosine 403 is
accessible from the external bulk medium. In contrast, transported
substrates significantly slowed the reaction, suggesting that
during the transport cycle residue 403 becomes occluded. On the other
hand, transportable substrates are not able to protect Y403C
transporters against N-ethylmaleimide, which is highly
permeant but unable to modify cysteine residues buried within membrane proteins. These results indicate that tyrosine 403 is alternately accessible from either side of the membrane, consistent with its role
as structural determinant of the potassium binding site.
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INTRODUCTION |
Glutamate transporters, located in plasma membranes of nerve and
glial cells, are instrumental in keeping the synaptic concentration of
the transmitter below neurotoxic levels (1-4). Moreover, together with
diffusion, they may help to terminate its action in synaptic transmission (5, 6). They achieve this by an electrogenic process
(7-9) where the transmitter is cotransported with three sodium ions
and a proton (10), followed by countertransport of a potassium ion
(10-13).
A glutamate transporter has been purified to near homogeneity and
reconstituted (14, 15). This transporter, termed GLT-1, has been cloned
(16), and its physiological importance is illustrated in knock-out
experiments (3, 4). Four other glutamate isotransporters have been
cloned (17-20) and the homology between these family members is around
50%.
Recently we identified two residues of GLT-1 important for potassium
coupling (13, 21). These residues, tyrosine 403 and glutamate 404, are
located in the most conserved region of the transporter. Nearby in the
primary sequence is aspartate 398, which is also critical for
transporter function (22). These and other observations, suggest that
this region may form part of the translocation pathway of the
transporter. The scanning cysteine accessibility method
(SCAM1) has permitted the
systematic identification of residues lining ion channels (for
instance, 23-25). We have therefore used this method to probe the
accessibility of residues 395-407. Strikingly, this study has led to
the identification of a conformationally sensitive residue. We suggest
that it is alternatingly accessible from either side of the membrane in
a substrate-dependent manner.
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EXPERIMENTAL PROCEDURES |
Cell Growth and Expression--
HeLa cells were cultured (26),
infected with recombinant vaccinia/T7 virus vTF7-3 (27), and
transfected with plasmid DNA encoding wild type or mutant GLT-1 (26).
Transport experiments with the GLT-1 substrate
D-[3H]aspartate were done as described (22).
Data presented are after subtracting the values obtained from cells
transfected with the vector, pBluescript SK
, alone. In
the case of some of the mutants and in particular if they were
subcloned into the cDNA encoding the cysteine-less GLT-1 (see
below), the sodium concentration of the transport medium was lowered to
15 mM (supplemented with 135 mM choline
chloride) as the sodium affinity of aspartate transport endogenous to
the HeLa cells is much lower than that of GLT-1 and its derivatives (data not shown). This enables us to obtain acceptable
signal/background ratios, at least 4-fold, even in the case of
relatively low activities exhibited by the cysteine-less transporter
and its derivatives.
Inhibition Studies with Sulfhydryl Reagents--
Prior to the
transport measurements, the cells, adhered to 24-well plates, were
washed with the 150 mM NaCl containing transport medium.
Each well was then incubated at room temperature with 200 µ l of this
solution (in case of different composition this is indicated in the
figure legends) and the indicated concentration of reagent under study.
After 5 min the medium was aspirated, and the cells were washed twice
with 1 ml of the transport solution. Subsequently they were assayed for
D-[3H]aspartate transport using solution to
which the labeled amino acid was added (22). The hydrophilic
methanethiosulfonate (MTS) reagents used were purchased from Toronto
Research Chemicals, Inc. Positively charged MTSEA and MTSET are
approximately 1 nm in length, differing only at the charged head group,
which measures 0.58 nm in MTSET and 0.36 nm in MTSEA (23). MTSES is
negatively charged and has an intermediate size (23). Whereas MTSEA is membrane permeant to some extent, the other two compounds are impermeant (28).
Site-directed Mutagenesis--
Mutagenesis (22, 29) was done
using uracil-containing single strand DNA derived from the shortened
GLT-1 clone (30) or its cysteine-less homologue. Most results reported
in this study concern individual replacements of selected residues of the wild type GLT-1 by cysteine, but cysteine mutants in a
cysteine-less background were also tested to confirm the specificity of
the effects observed in the wild type background. The cysteine-less GLT-1 was prepared and characterized as follows: Cysteines 536, 549 and
562 are clustered together at the carboxyl-terminal tail. Because we
have evidence that the major part of this tail is not required for
transport,2 the stretch from
residue 533 to residue 563 was deleted by site-directed mutagenesis. At
the same cycle of the mutagenesis another primer was annealed
simultaneously to the uracil-containing single-stranded DNA. It was
designed to convert cysteine 38 to serine. After verifying by DNA
sequencing that these changes were indeed incorporated, the construct
was expressed and shown to be unimpaired for
D-[3H]aspartate transport. Subsequently
uracil-containing single-stranded DNA was prepared from this construct,
and new cycles of mutagenesis, sequencing, and expression were
performed to mutate the remaining cysteines to serine. In the second
cycle, cysteines 60, 293, and 296 were converted, in the third 184, and
in the fourth 373. In the subsequent cycle a deca-histidine tail was
introduced between the last residue of GLT-1, lysine 573, and the stop
codon. A major reason to prepare the above construct was for the
determination of the membrane topology of GLT-1 by one of several
approaches,3 namely,
biotinylation of single cysteine-containing transporters. In this
approach artifactual biotinylated bands are observed running with a
similar mobility as the above described construct. To detect transporter-related signals, it was necessary to "mass-tag" the construct so that the transporter band would be resolved from the
artifactual ones. We have shown previously that most of the amino-terminal tail of the GABA transporter GAT-1 is not required for
its functional expression (31). Therefore, we have inserted upstream of
the original first methionine the coding sequence for the first 41 amino acids of GAT-1 preceded by the DNA sequence 5'-ATGCATTTCGTGCTCCGAGAC-3', encoding for the amino acids MHFVLRD; the
latter was added to facilitate its insertion using standard molecular
biology approaches. Using GAT-1 as a template, the primers used for
amplification were designed such that on both ends of the amplified
fragment a StuI site was created. After digestion with
StuI, the fragment was ligated into blunt ends created in the cysteine-less histidine-tagged GLT-1 described above, by cutting it
with PflMI and removal of the 3'-overhangs with bacteriophage T4 DNA polymerase. Plasmid DNA of several of the
transformants was isolated and one of those, which gave rise to
aspartate transport after expression, was retained. The result is that
the 48 amino acids are inserted immediately amino-terminal to the
original start codon of GLT-1. All the expected features of the
modified cysteine-less GLT-1 were verified by sequencing in both
directions and upon expression its
D-[3H]aspartate transport activity was found
to be 44.5 ± 1.5% of that of the wild type GLT-1
(n = 7), and the level of expression as estimated by
immunoprecipitation was comparable to that of the wild type (data not
shown). The characteristics of transport of this cysteine-less
histidine-tagged GLT-1 transporter were very similar to that of the
wild type GLT-1, as judged by affinity to D-aspartate (Fig.
1A) and sodium (Fig.
1B). Single cysteine-containing transporters were made using
uracil-containing single strand DNA derived from this construct.
Mutants were verified by DNA sequencing. Mutations Y403C and E404C were
subcloned into wild type or cysteine-less GLT-1 using the enzymes
BsrGI and BstEII. Subcloned DNAs were sequenced
in both directions between these restriction sites.

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Fig. 1.
Characteristics of
D-[3H]aspartate uptake by the cysteine-less
GLT-1. HeLa cells were infected with the recombinant vaccinia/T7
virus and transfected with cDNA of the wild type or the
cysteine-less GLT-1. After washing the cells with 150 mM
choline chloride medium transport of
D-[3H]aspartate was measured as described
under "Experimental Procedures." The influx solutions were
supplemented with the indicated concentrations of unlabeled
D-aspartate (A). In B the 150 mM NaCl medium was used as the 100% value, and the sodium
concentration was varied as indicated using choline chloride to
supplement the chloride salt concentration to 150 mM.
Values are the average (± S.E.) of triplicate determinations and are
expressed as percent activity of wild type or cysteine-less GLT-1
measured in the presence of 150 mM NaCl in the absence of
unlabeled D-aspartate. , GLT-1; , cysteine-less
GLT-1.
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RESULTS |
Cysteine-scanning Accessibility of Residues 395-407--
Residues
395-407 of the glutamate transporter GLT-1 have been changed one at a
time to cysteine. After transient expression of the wild type and the
cysteine replacement mutants in HeLa cells, transport of
D-[3H]aspartate was monitored.
No transport can be detected when cysteine is introduced in each of the
five consecutive residues from 396 to 400 (Fig.
2). The possible role of these residues
in the transport process will be addressed under "Discussion."
Right now we focus on the remaining residues, where cysteine
replacement leaves significant biological activity (Fig. 2).

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Fig. 2.
Sodium-dependent
D-[3H]aspartate uptake in HeLa cells
expressing wild type and mutant transporters. Uptake was measured
as described under "Experimental Procedures." Values are average
(± S.E.)of triplicate determinations and are expressed as percent of
wild type values.
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Transport activity of wild type GLT-1 is not inhibited by 2.5 mM of the MTS derivative (MTSEA), and the same is true for
all the active cysteine replacement mutants with the exception of Y403C
(Fig. 3). When the preincubation with the
sulfhydryl reagent is carried out in a sodium-free medium (choline
substitution) E404C transporters become sensitive to the reagent as
well (Fig. 4). It appears that sodium
protects against covalent modification at this position, since similar
data are obtained when the E404C mutation is introduced in a
cysteine-less GLT-1 background (data not shown). Although not visible
in the experiments depicted in Figs. 3 and 4, there is also a partial
protection of Y403C transporters by sodium against inhibition by MTSEA.
This can be readily observed at lower concentrations of this sulfhydryl
reagent (Fig. 5). Similar results have
been observed with the Y403C mutation introduced in a cysteine-less
background (data not shown, but see Fig. 9). Other cations are not
effective except for lithium, which affords a small but significant
protection (data not shown). It is of interest to note that also in the
case of E404C, lithium is the only cation besides sodium that can give
a significant protection against inhibition by MTSEA (data not
shown).

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Fig. 3.
Inhibition of transport by MTSEA in the
presence of sodium. HeLa cells, expressing wild type or the
indicated mutant transporters, were washed with the standard NaCl
medium and subsequently preincubated in the same medium supplemented
with 2.5 mM MTSEA. After 5 min at room temperature the
cells were washed and assayed for
D-[3H]aspartate transport as described under
"Experimental Procedures."
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Fig. 4.
Inhibition of transport by MTSEA in the
absence of sodium. This was carried out exactly as described in
the legend to Fig. 3, except that in the preincubation and washing
solutions sodium was replaced by the same concentration of
choline.
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Fig. 5.
Effect of sodium on inhibition of the Y403C
transporter by MTSEA. HeLa cells expressing the transporters were
preincubated for 5 min in sodium- or choline chloride-containing media
without (control) or with 0.25 mM MTSEA. After washing the
cells, sodium-dependent uptake of
D-[3H]aspartate was measured in triplicate in
a 15 mM NaCl + 135 mM choline
chloride-containing medium. Data are presented as percent activity of
the control (± S.E.) of each condition.
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Characterization of the Inhibition of Y403C by MTS
Reagents--
As will be shown below, the most striking observation
described in this paper is the opposite effect of transportable and nontransportable substrates on the inhibition of Y403C by MTS reagents
in a sodium-containing medium. In contrast, E404C transporters are not
inhibited very much by MTSEA in the presence of sodium (Fig. 3), and
the same is true when transporter substrates are added (data not
shown). Therefore, we have focused on the characterization of
functional effects of covalent modification of Y403C transporters. Wild
type GLT-1 is not inhibited by the positively charged MTSEA and MTSET
(Figs. 3, 4, and 6A) and also
not by MTSES (data not shown). In contrast with wild type, not only
MTSEA (Fig. 3) but also MTSET potently inhibit Y403C transporters (Fig.
6A). On the other hand, negatively charged MTSES does not
(Fig. 6B). Because MTSES is intermediate in size between
MTSEA and MTSET (23), it appears that the positive charge in the
reagent determines its ability to inhibit. After preincubation of Y403C
transporters with MTSES, they still can be inhibited by a subsequent
exposure to MTSEA (Fig. 6B). This speaks against the
possibility that the residue can be covalently modified MTSES but that
this does not lead to inhibition of transport.

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Fig. 6.
Effect of other MTS reagents on Y403C
transporters. HeLa cells expressing wild type or Y403C
transporters were preincubated in the standard NaCl medium containing
2.5 mM MTSEA
( ) or 1 mM
MTSET ( ) (A)
or 10 mM MTSES (B) for 5 min. In B an
additional 5 min of incubation with 2.5 mM MTSEA was
carried out. Subsequently transport was measured using the 15 mM NaCl + 135 mM choline chloride-containing
medium. , control; WT, wild type.
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In the experiment depicted in Fig. 7,
Y403C transporters are preincubated with 0.25 mM MTSEA in a
sodium-containing medium. When transport of
D-[3H]aspartate is subsequently measured, an
inhibition of almost 80% is observed. When a saturating concentration
(1 mM) of transportable acidic amino acids such as
D- and L-aspartate or L-glutamate
is added during the preincubation, this causes a marked protection of
the transporters against the inhibition by MTSEA (Fig. 7). This
protection is only observed in the presence of sodium ions (data not
shown). Amino acids that are not substrates of GLT-1, such as GABA or
glycine, do not protect (Fig. 7). The protective effect of
D-aspartate is dose dependent; half-maximal effects are
observed with 30-50 µM (data not shown), which is in
good agreement with the apparent Km of
D-aspartate transport in HeLa cells expressing GLT-1 (22).
The protection by D-aspartate is observed at all MTSEA
concentrations examined (Fig.
8A). The substrate similarly
affords protection against the impermeant MTSET (Fig. 8B).
It is also apparent that the larger MTSET inhibits less potently than
MTSEA, presumably because of steric constraints. Again, inhibition by
the MTS reagents and the protection by transportable substrates are the
consequence of a direct modification of the cysteine at the 403 position, because similar results are obtained with transporters in
which this is the only cysteine (Fig. 9). Strikingly, the competitive inhibitor dihydrokainate (12, 32), a
nontransportable glutamate analogue (32-34), does not protect, and in
fact even potentiates the inhibition by the MTS reagents. Thus
transportable substrates induce a conformational change making the
residue less accessible to the impermeant reagent, whereas the blocker
seems to lock the transporter in a conformation where it is more
accessible to the extracellular bulk medium. Importantly, the wild type
as well as the cysteine-less GLT-1 are not inhibited by MTSEA and MTSET
even in the simultaneous presence of dihydrokainate (data not
shown).

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Fig. 7.
Effect of amino acids on inhibition of Y403C
transporters by MTSEA. HeLa cells expressing Y403C were incubated
in the standard medium in the presence and absence of 0.25 mM MTSEA ( )
and the indicated amino acids (at 1 mM final
concentrations). After washing, transport in the low sodium medium was
measured, and the data are expressed as percent activity remaining
relative to the respective control without MTSEA. , control.
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Fig. 8.
Effect of D-aspartate and
dihydrokainic acid on inhibition of transport of Y403C by MTSEA and
MTSET. Cells expressing Y403C transporters were preincubated under
standard conditions with the indicated concentrations of MTSEA or MTSET
in the absence ( ) or presence of 1 mM of either
dihydrokainate ( ) or D-aspartate ( ). Uptake was
measured in the low sodium-containing medium.
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Fig. 9.
Effect of D-aspartate and
dihydrokainic acid on inhibition of transport of Y403C in a
cysteine-less background. The conditions were as described in the
legend to Fig. 8 using MTSEA at 0.1 mM. It should be noted
that here, in contrast, to Fig. 8, data are presented as % inhibition.
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Effects of NEM--
The conformational change induced by aspartate
may cause burial of the 403 residue within the core of the transporter.
Alternatively, it may reflect the transport step itself, and the
residue may become accessible to the inside bulk medium. In the former
case the residue is not expected to be reachable by NEM, which
permeates lipid bilayers readily but is unable to modify cysteine
residues within transmembrane-spanning
helices (35, 36). The
maleimide does not inhibit wild type GLT-1 (data not shown) but
inhibits the activity of the Y403C transporter (Fig.
10). In contrast with its protection
against MTS reagents, D-aspartate does not protect the
Y403C transporters against NEM (Fig. 10). This is consistent with the
idea that its protection against relatively impermeant MTS reagents is
because of the movement of the 403 residue from the outside of the cell
to the inside. Similar results are obtained with the mutation
introduced in a cysteine-less background (data not shown). The partial
protection observed with dihydrokainate will be discussed below.

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Fig. 10.
Effect of D-aspartate and
dihydrokainic acid on the inhibition of transport of Y403C by
N-ethylmaleimide. HeLa cells expressing Y403C were
incubated with the indicated concentrations of
N-ethylmaleimide (NEM) in the presence or absence
of 1 mM of either D-aspartate or dihydrokainic
acid. Transport was done in the low sodium-containing medium.
Con, control; dAsp, D-aspartate;
DHK, dihydrokainic acid.
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DISCUSSION |
Analysis of amino acid residues 395-407 of GLT-1 by SCAM leads to
two major conclusions: 1) Within the putative pore-forming domain of
GLT-1 are five consecutive residues (396-400) where mutation to
cysteine leads to complete loss of function, a result consistent with
the structural conservation and functional importance of this region.
2) Residue 403, which has been previously shown to interact with
cotransported cations (21), behaves as if it is alternately accessible
from either side of the membrane in such a way that glutamate
transport promotes its exposure to the inside, whereas binding of a
nontransportable analog promotes its exposure to the outside. This
residue is located in the middle of transmembrane
-helix seven
(37).3
One of the five residues that do not tolerate replacement by cysteine
(Fig. 2) is aspartate 398. Its replacement by either asparagine,
glycine, or glutamate also leads to loss of function, and this is not
because of a defective biosynthesis or targeting (22). Injection of
D398E cRNA into Xenopus oocytes results in neither glutamate
transport currents nor transient currents reflecting sodium
binding.4 It is possible
therefore that this residue, together with asparagine 396, methionine
397, glycine 399, and threonine 400, plays a crucial role in sodium
binding. Recent experiments in our laboratory indicate that sodium and
potassium binding sites are close to each other (21). Glutamate 404 and
tyrosine 403 are involved in potassium binding (13, 21), and residues
396-400 are in fact nearby. Consistent with this is the protection of
E404C and Y403C transporters by sodium against thiol modification
(Figs. 3-5). This modification occurs at these very positions, as
evidenced by the fact that the same results are also obtained in a
cysteine-less background. This rules out a scenario in which the
introduction of a new cysteine residue induces a change in the
transporter's structure such that modification now may occur at one of
the nine endogenous cysteines of GLT-1. The cysteine-less GLT-1
transporter has around 40% of the wild type activity, and therefore
most of the experiments have been done in the wild type background.
However, they have been validated in the cysteine-less background as
shown, for instance, in Fig. 9. It is of interest to note that the only
ion that can partially substitute for sodium in the protection
experiments is lithium. This is not unexpected as only this cation can
replace some of the three sodium ions in the transport process (38). The fact that only positively charged MTS reagents inhibit at the 403 position (Fig. 6), also supports the idea that it is part of a
negatively charged binding pocket for cations, which repels the
negatively charged MTSES.
On the other hand, the protection by transportable acidic amino acids
against inhibition by positively charged MTS reagents (Figs. 7-9) is
because of a conformational change induced by their binding at a
distinct site. This is inferred because the opposite effect is induced
by binding to this site of the nontransportable dihydrokainate, which
locks the transporter in a conformation where tyrosine 403 is
accessible from the external bulk medium. Transportable substrates
cause a conformational change where this residue becomes occluded.
Further evidence for a close association of this phenomenon with the
transport step itself is that it was only observed in the presence of
the cosubstrate sodium. The sensitivity to NEM during transport
conditions (Fig. 10), in contrast to the protection against MTS
reagents (Figs. 8 and 9), suggests that now tyrosine 403 becomes
accessible to the aqueous intracellular compartment.
Determinants of the binding site for the acidic amino acids are located
on a stretch of 76 amino acid residues (39), which also contains the
cation binding site (13, 21). One explanation for the partial
protection by dihydrokainate against NEM may be that the former is
bound close enough to hamper the access of NEM, which is bulkier than
the MTS reagents.
Tyrosine 403 is involved in the interaction of the transporter with
internal as well as external potassium (21) and it appears, together
with glutamate 404 (13), to be a structural determinant of the
potassium site. Because of the role of potassium, which is to promote
the return of the unloaded transporter to the side of the membrane from
where substrate can be moved (11-13), it is to be anticipated that
during the transport cycle the potassium binding site should be
alternately accessible to either side of the membrane. The findings
documented here are entirely consistent with this. We anticipate that
application of SCAM to other interesting regions of GLT-1 and to other
transporters, may lead to important structural and functional insights
as well.