(Received for publication, April 19, 1995)
From the
Sodium-coupled glutamate transporters, located in the plasma
membrane of nerve terminals and glial processes, serve to keep its
extracellular glutamate concentration below extracellular levels.
Moreover, they help in conjunction with diffusion to terminate the
transmitter's action in synaptic transmission. We have
investigated the role of negatively charged amino acid residues of
GLT-1, a cloned (Na
L-Glutamate is a major excitatory neurotransmitter in
the mammalian central nervous system (Collingridge and Lester, 1989).
However, at elevated extracellular concentrations, L-glutamate
is neurotoxic (Choi, 1988). Electrogenic (Na
A small gene family that mediates the
uptake of acidic amino acids has recently been defined by molecular
cloning (reviewed in Kanner(1993), Kanai et al.(1993), and
Amara and Arriza(1993)). Three related glutamate transporters (
The
stoichiometry of the transport process is likely to be two sodium ions
accompanying each glutamate anion, whereas one potassium and one
hydroxyl ion are transported in the opposite direction (Kanner and
Sharon, 1978; Stallcup et al., 1979; Bouvier et al.,
1992). Because all the substrates are charged molecules, conserved
charged amino acids located in the hydrophobic domain of the glutamate
transporters are likely to be important for the binding and
translocation of the substrates. Structure predictions indicate the
existence of six transmembrane
The following Primers were used to make the indicated
mutations: D398N, 5`-AAAGGGCTGTACCATTCATGTTAATGGTTGC-3`; D398E and
D398G, 5`-GTAAAGGGCTGTACCC(C/T)CCATGTTAATGGTTG-3`; E404N,
5`-GAAGATGGCTGCCACGGCGTTGTAAAGGGCTGTACCATC-3`; E404D and E404G,
5`-GAAGATGGCTGCCACGGCG(T/C)CGTAAAGGGCTGTACCATC-3`; E461N,
5`-CACCAGCAGACTGATGTCGTTTGTCGGCAGGCCCACAGC-3`; D462N,
5`-TGCCACCAGCAGACTGATGTTCTCTGTCGGCAGGCCCAC-3`; D470N,
5`-TATCCAGCAGCCAGTTCACTGCCACCAGCAG-3`; and D470E and D470G,
5`-TCTATCCAGCAGCCAC(C/T)CCACTGCCACCAGCA-3`. Mutations were confirmed by
DNA sequencing and subcloned into wild type.
For the mutations
E404D, E404G, E404N, D398N, D398G, and D398E, the enzymes HindIII, Asp700, and BstEII were used. Two
fragments were taken from the wild type; the first was a
3.35-kb
For the mutations D470N, D470E, and D470G,
we used the enzymes PstI and BstEII. The 4.45-kb
fragment from the wild type was ligated with the 0.35-kb fragment from
the mutants. The subcloned cDNAs were sequenced from both directions
between the sites of the indicated restriction enzymes.
Fig. 1shows [
Figure 1:
L-[
Figure 2:
L-[
Figure 3:
Immunoprecipitation of wild type and
mutant proteins synthesized in Hela cells. Hela cells were infected
with recombinant vaccinia/T7 virus and transfected with pBluescript
containing the wild type or the indicated mutants. Cells were labeled
with [
Figure 4:
L-[
Figure 5:
L-[
Figure 6:
Uptake of the stereoisomers of
[
Figure 7:
Inhibition of D-[
In this study we have investigated the role of negatively
charged amino acids of GLT-1 on its function. We have limited ourselves
to those residues that are located in hydrophobic surroundings and that
are highly conserved in the three presently known glutamate
transporters. They are all located on a highly conserved stretch of 72
amino acids in the carboxyl-terminal part of the transporter. The
predicted topology of this stretch is controversial (Kanai and Hediger,
1992; Storck et al., 1992; Pines et al., 1992) and
has not yet been resolved experimentally. Out of the five amino acid
residues examined, three, Asp 398, Asp 470, and Glu 404, have been
found to be critical for the function of the transporter ( Fig. 1and 2). It appears that these three residues are important
for the activity of the transporter rather than its stability (Fig. 3) and its translocation to the plasma membrane (Fig. 4). It is unlikely that one of these residues is involved
in the formation of an ion pair with histidine 326. This residue,
previously identified as critical for the function of GLT-1 (Zhang et al., 1994), is located in putative transmembrane helix 6.
In the lactose transporter of E. coli, pairs of positive and
negative amino acids have been identified. Whereas each of these
residues by itself appeared critical, the double mutants regained
activity (King et al., 1991; Sahin-Toth et al., 1992;
Dunten et al., 1993; Sahin-Toth and Kaback, 1993). Double
mutants, in which histidine 326 and either of the above residues were
substituted by asparagine, did not regain activity (Fig. 5) even
though full-length transporters were synthesized (Fig. 3).
Although these findings do not support the concept of ion pair
formation between any of the negatively charged residues with histidine
326, it is at the present time not possible to rule it out altogether.
Right now we have no evidence on the role of residues Asp 398 and
Asp 470 in the functioning of the transporter. It is not sufficient
that they are merely negatively charged, because they cannot be
replaced even by glutamate (Fig. 2). Therefore, the interaction
that these amino acid residues undergo, either with other residues of
the transporter or with one or more of the substrates, must be rather
specific. Because both aspartate residues are located in a hydrophobic
environment, it is tempting to speculate that they could be involved in
the binding and/or translocation of one or more of the cotranslocated
ion species, such as sodium. In fact, although the stoichiometry has
not yet been established definitively, it is likely that two sodium
ions are translocated per glutamate (Kanner and Sharon, 1978; Stallcup et al., 1979; Bouvier et al., 1992). Further evidence
for this idea could be obtained by measuring partial reactions
involving sodium binding. In fact, a recent report indicates that in
the human counterpart of GLT-1, sodium transients could be observed
that are dihydrokainate-suppressible (Wadiche et al., 1995).
Residue Glu 404, on the other hand, is clearly not involved in the
binding of the cotranslocated ion species. Even though glutamate
transport is severely impaired when the residue is mutated to
asparagine, glycine, or aspartate ( Fig. 1and Fig. 2),
very significant sodium-dependent transport of the alternative
substrates L- and D-aspartate is observed in each of
these mutants (up to 80% for E404D of wild type D-aspartate
transport rates) (Fig. 6). Thus, the substrate specificity of
GLT-1 transporters when mutated at the Glu 404 position is altered. It
is of course impossible to rule out the indirect effects of the mutated
amino acid due to a conformational change transmitted from a distant
site, like with any other site-directed mutagenesis study. We feel this
is unlikely because high levels of D- and L-aspartate
transport are observed in the Glu 404 mutants. Furthermore, the residue
is located in the area of the transporter that is almost invariant in
the three known glutamate transporters EAAC-1, GLAST, and GLT-1. Out of
33 residues, 31 are identical in all three and two are conservatively
substituted.
Even though glutamate is poorly transported in the
E404D mutant (Fig. 2), it clearly is capable of binding to the
transporter (Fig. 7C). The same is true for
dihydrokainate (Fig. 7B) non-transportable glutamate
analogue (Pocock et al., 1988; Barbour et al., 1991;
Arriza et al., 1994). The differential inhibition of glutamate
transport relative to aspartate therefore seems to be associated with a
defect in its translocation or its dissociation from the transporter on
the inside. With regard to substrate translocation, it is of interest
to mention that we have recently found evidence for two glutamate-based
transporter states. After the initial binding it undergoes a
conformational change that represents or is tightly associated with the
transport step. Nevertheless, the mutation of the Glu 404 residue has
some effect on substrate binding because D-aspartate (Fig. 7A) and L-aspartate (data not shown)
exhibit increased affinity to the E404D transporter. All of the aspects
discussed here are interrelated and point to location of Glu404 in the
vicinity of the glutamate-aspartate permeation pathway. It is
anticipated that study of other amino acid residues located in this
highly conserved part of the transporter will give us further insights
into the molecular events of the translocation of glutamate.
We thank Dr. Bernard Moss for the provision of
recombinant virus vTF7-3 and Beryl Levene for expert secretarial
help.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
+ K
)-coupled
glutamate transporter from rat brain. Using site-directed mutagenesis
we modified these negative residues, which are located in hydrophobic
surroundings and are highly conserved within the glutamate transporter
family. Out of five residues meeting these criteria, three, aspartate
398, glutamate 404, and aspartate 470, are critical for heterologously
expressed glutamate transport. This defective transport cannot be
attributed to the mere requirement of a negative charge at these
positions. After prelabeling of the proteins with
[
S]methionine, immunoprecipitation of all mutant
transporters indicates that their expression levels are similar to that
of wild type. No cryptic activity was revealed by reconstitution
experiments aimed to monitor the activity of transporter molecules not
located in the plasma membrane. Significantly, whereas all of the
mutants at the glutamate 404 position exhibit impaired transport of
glutamate, they possess considerable transport of D- and L-aspartate, up to 80% of wild type values. Binding of
glutamate is not impaired in these mutants. Our observations indicate
that the glutamate 404 residue may be located in the vicinity of the
glutamate-aspartate permeation pathway.
+
K
)-coupled L-glutamate transporters safeguard
against this danger by rapidly removing the transmitter from the
extracellular fluid (Kanner and Schuldiner, 1987; Nicholls and Attwell,
1990). Furthermore, they may help, together with diffusion, to
terminate its action in synaptic transmission (Mennerick and Zorumski,
1994; Tong and Jahr, 1994).
55%
homology) have been cloned from rat brain: GLAST (Storck et
al., 1992), EAAC-1 (Kanai and Hediger, 1992), and GLT-1 (Pines et al., 1992). These three glutamate transporters form a small
family that also includes a sodium-coupled small neutral amino acid
transporter (Arriza et al., 1993; Shafqat et al.,
1993) as well as bacterial dicarboxylic acid and proton-coupled
glutamate transporters (Jiang et al., 1989; Tolner et
al., 1992). The GLT-1 protein has been purified to near
homogeneity and reconstituted (Danbolt et al., 1990, 1992). It
represents around 0.6% of the protein of crude synaptosomal fractions
(Danbolt et al., 1990) and is exclusively located in the fine
astroglial process of rat brain (Danbolt et al., 1992). The
cloning and characterization of the human homologues of these proteins
have recently been described (Arriza et al., 1994).
-helices as well as a hydrophobic
domain, which is located in the carboxyl-terminal half of the
transporters. The number of transmembrane segments in this domain is
controversial (Kanai and Hediger, 1992; Pines et al., 1992;
Storck et al., 1992). In the six predicted
-helices, two
positive conserved amino acid residues are found. One of these two,
histidine 326 (GLT-1 numbering), was found to be critical for transport
activity and was implicated in the proton translocation accompanying
sodium- and potassium-coupled glutamate transport (Zhang et
al., 1994). Five negatively charged, highly conserved amino acid
residues are located in the hydrophobic domain close to the carboxyl
terminus. We have studied the role of these residues using
site-directed mutagenesis and find that three of them are critical. One
of these, glutamate 404, appears to be involved in the determination of
the amino acid substrate specificity.
Methods
Cell Growth and Expression
HeLa cells were
cultured in Dulbecco's modified Eagle's medium supplemented
with 10% fetal calf serum, 200 units/ml penicillin, 200 µg/ml
streptomycin, and 2 mM glutamine. Infection with recombinant
vaccinia/T7 virus vTF7-3 and subsequent transfection with plasmid
DNA were done as described (Keynan et al., 1992). Amino acid
transport in intact cells and reconstituted systems was done as
described (Pines et al., 1992). Results are usually given as
the percentage of transport of the indicated mutant relative to the
wild type, each after subtraction of the value of vector alone. This
correction factor in intact cells was around 25% of the gross counts of
the wild type for L-[H]glutamate and
around 5% for either stereoisomer of
[
H]aspartate. It was negligible for transport in
reconstituted systems. In all cases, this transport was fully
sodium-dependent. These results are entirely consistent with recent
work on the endogenous acidic amino transport in the HeLa cells (Igo
and Ash, 1995). Immunoprecipitation was performed as published (Pines et al., 1992), except that the lysate was shaken end over end
at 4 °C for 1 h with protein A-Sepharose CL-4B beads that had been
incubated with preimmune serum. After removal of these beads by
centrifugation, the antibody was added, and after 2 h the described
protocol was followed. Protein was determined on whole cells (Bradford,
1976) or on reconstituted proteoliposomes (Peterson, 1977) as
described. Polyacrylamide gel electrophoresis was as described
(Laemmli, 1970) using 4% stacking and 10% separating gels. Size
standards (Pharmacia Biotech Inc.) were run in parallel and visualized
by Coomassie Blue staining. Antibody against the whole transporter was
raised as described (Danbolt et al., 1992).
Site-directed Mutagenesis
Mutagenesis was
performed as described (Kunkel et al., 1987). The shortened
GLT-1 clone (Casado et al., 1993) was used to transform Escherichia coli CJ236 to ampicillin resistance. From one of
the transformants single-stranded uracil containing DNA was isolated
upon growth in a uridine-containing medium according to the standard
protocol from Stratagene, using helper phage R408. This yields the
sense strand, and consequently the mutagenic primers were designed to
be antisense.
(
)BstEII-HindIII
fragment that was dephosphorylated with calf intestine phosphatase, and
the second was a 1.2-kb HindIII-Asp700 fragment. The
two fragments were ligated with 0.25-kb Asp700-BstEII
fragments of the mutants.
Materials
Polynucleotide kinase, DNA polymerase, and DNA ligase (all
from T) and phosphatase from calf intestine were from
Boehringer Mannheim. Restriction enzymes were from New England Biolabs
and Boehringer Mannheim. Sequenase kits (version 2.0) were from U. S.
Biochemical Corp. D-
S-
ATP (1,000 Ci/mmol)
and [
S]methionine (1,000 Ci/mmol) were from
Amersham Corp. L-[
H]Aspartate (15.5
Ci/mmol) and D-[
H]aspartate (11.5
Ci/mmol) were from DuPont NEN. [
H]Glutamate (60
Ci/mmol) was from American Radiolabelled Chemicals, Inc. The tissue
culture medium, serum, penicillin/streptomycin, and L-glutamine were from Biological Industries (Kibbutz Bet
Ha'Emek, Israel). Transfection reagent (N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium
methyl sulfate) was from Boehringer Mannheim. The vaccinia/T7
recombinant virus was a gift from Dr. Bernard Moss (National Institutes
of Health). Brain lipids were prepared from bovine brain as published
(Folch et al., 1957). Protein A-Sepharose CL-4B, asolectin (P
5638, type II-S), valinomycin, uridine, cholic acid, and all other
materials were purchased from Sigma.
H]glutamate
transport by cells expressing of wild type GLT-1 or mutant
transporters. The five negatively charged and highly conserved residues
located in the hydrophobic domain close to the carboxyl terminus have
each been mutated to asparagine. After the mutations were verified by
sequencing, the mutant cDNAs were expressed in HeLa cells using the
recombinant vaccinia/T7 virus (Fuerst et al., 1986) as
described (Keynan et al., 1992). Although E461N and D462N
transporters exhibit transport activity similar to that of the wild
type, the D398N, E404N, and D470N mutants are severely impaired. The
cDNAs carrying these three mutants as well as that of the wild type
were cut with the appropriate restriction enzymes (see
``Experimental Procedures'') so that a relatively small
fragment of the mutant cDNAs could be subcloned into that of the wild
type. These inserted fragments were sequenced in both directions and
found to be unchanged with regard to the wild type, except for the
mutation itself. Upon expression of these mutants, the same low levels
of glutamate uptake as shown in Fig. 1were found (data not
shown). These results indicate that the three mutated residues may be
important for the function of the glutamate transporter GLT-1. In order
to find out whether asparagine at these positions affects functional
expression, each of them was mutated to glycine as well as to the
alternative negatively charged amino acid. These mutations were
subcloned and verified by sequencing as above. The results of
expression experiments with the mutant cDNAs are summarized in Fig. 2. The results indicate that the amino acid residues
aspartate 398, glutamate 404, and aspartate 470 themselves are
important for the functional expression of the GLT-1 transporter.
Because replacements of aspartate by glutamate and vice versa do not
yield transporters with high activity (Fig. 2), it is clear that
it is not sufficient to merely have a negative charge at these
positions. It is of interest to note that, although the activity of
transporters bearing mutations in Asp 398 or Asp 470 is undetectable, a
small but significant activity is observed in mutants at the Glu 404
position. The highest activity (18-19% of wild type) is observed
when the residue is replaced by aspartate (Fig. 2).
H]Glutamate
uptake by wild type and asparagine replacement mutants. Hela cells were
infected with recombinant vaccinia/T7 virus and transfected with cDNA
of the vector alone, the wild type (W.T), or the indicated
mutants. Results are given as percentages of transport of the indicated
mutants relative to the wild type as described under
``Experimental Procedures.'' Each bar is the mean
± S.E. of four different
experiments.
H]Glutamate
uptake of mutants at positions Asp 398, Glu 404, and Asp 470. Hela
cells were infected with recombinant vaccinia/T7 virus and transfected
with cDNA of the vector alone or the wild type (W.T) or the
indicated mutants. Each bar is the mean ± S.E. of
3-6 different experiments.
The
impaired transport of the mutants is not due to lower transporter
levels. To determine this, wild type and mutant transporters are
expressed in HeLa cells labeled with
[S]methionine and immunoprecipitated by an
antibody raised against the purified GLT-1 transporter (Danbolt et
al., 1990, 1992). The wild type transporter runs as a doublet
around 67 kDa (Fig. 3, lane 1) and is not observed in
HeLa cells expressing the vector alone (Fig. 3, lane
2). The same is true with the higher molecular weight bands, which
probably represent dimers and trimers of the transporter. It is well
known that the transporter has a strong tendency to aggregate and that,
once formed, these aggregates are not disrupted under the conditions of
SDS-polyacrylamide electrophoresis (Danbolt et al., 1992). The
pattern is very similar to that observed with an antibody raised
against a sequence from the carboxyl-terminal end of GLT-1 (Zhang et al., 1994). All of the mutant transporters at the Asp 398,
Glu 404, and Asp 470 positions are present at levels similar to the
wild type, as exemplified in several of the mutants (Fig. 3, lanes 3-7).
S]methionine, lysed, and
immunoprecipitated with antibody against whole transporter as described
under ``Experimental Procedures''. Lane 1, wild
type; lane 2, vector alone (pBluescript SK
); lanes 3-7, mutants D398N, D470N, E404N, E404G, and
E404D, respectively; lanes 8-10, double mutants
H326N,D398N, H326N,D470N, and N326N-E404N,
respectively.
Because the mutants still produce normal
transporter levels, it is possible that they are inefficiently targeted
to the plasma membrane. One would expect that cells expressing a mutant
transporter that is intrinsically active but is inefficiently targeted
to the plasma membrane would have a cryptic transport activity.
Detergent extraction of the cells expressing such transporters followed
by reconstitution of the solubilized proteins is likely to yield
transport activity even if they were originally residing in internal
membranes. In fact, such cryptic transport activity has been observed
using this assay with some mutants of the GABA transporter GAT-1
(Kleinberger-Doron and Kanner, 1994) and GLT-1 (Zhang et al.,
1994). In this series of experiments, the glutamate transport in the
wild type ranges from 2-5 pmol/min/mg protein. This is similar to
the values observed in whole cells. This activity is completely
sodium-dependent and not observed with cells expressing the vector
clone (data not shown). Upon solubilization, no cryptic transport
activity is revealed in any of the mutants at the three positions (Fig. 4).
H]Glutamate
uptake in proteoliposomes containing wild type or mutant transporters.
Hela cells were infected with vaccinia/T7 virus and transfected with
cDNA of the wild type (W.T) or the indicated mutants. The
cells were treated with cholate, and the solubilized proteins were
reconstituted with asolectin/brain lipids using spin columns as
described under ``Experimental Procedures.'' The results are
the averages ± S.E. of transport of 3-6 experiments.
10-30 µg of protein were used for each transport
reaction.
It is in principle possible that one of the
negatively charged amino acids stabilizes the transporter by
neutralizing the critical histidine 326 (Zhang et al., 1994).
Evidence for such an idea would be that double mutants in which both
the histidine as well as one of the negatively charged amino acids are
replaced would regain activity. This is not the case (Fig. 5),
even though in each of these cases the transporter is synthesized (Fig. 3, lanes 8-10). Neither did these doubly
mutated transporters regain activity upon solubilization and
reconstitution (data not shown).
H]Glutamate
uptake by double mutants. Hela cells were infected with recombinant
vaccinia/T7 virus and transfected with cDNA of the wild type (W.T), the indicated double mutants (H326N-D398N, H326N-E404N, and H326N-D470N), or the vector alone.
Results are given as percentages of transport of the indicated mutant
relative to the wild type. Each bar is the mean ± S.E.
of 3-5 different experiments.
In view of the above, it appears
that Asp 398, Glu 404, and Asp 470 are required for the intrinsic
activity of GLT-1. Suitable assays to measure binding of the positively
charged cosubstrates sodium and potassium are presently lacking.
Therefore, at the present time, we cannot directly provide evidence for
the involvement of one or more of the three negatively charged residues
in these functions. However, glutamate itself also has a positive
charge: its amino group. Furthermore, we know that its binding pocket
must exhibit some flexibility. Even though the glutamate transporters
are stereospecific, preferring L-glutamate over its D-counterpart, there is very little discrimination between the D- and L-isomers of aspartate. Both stereoisomers are
excellent substrates for the transporter. Interestingly, whereas D398E
and D470E transporters are completely inactive with both D-
and L-aspartate, this is not the case with E404D (Fig. 6, A and B). Even though this mutant
transporter is greatly impaired in glutamate translocation ( Fig. 1and Fig. 2), considerable transport of D-
and L-aspartate are catalyzed by it (Fig. 6, A and B). In the case of D-aspartate, activity was
almost the same as that of the wild type. Considerable activity, albeit
less than that of E404D, is also observed with E404G and E404N
transporters (Fig. 6, A and B). Even though
glutamate transport by the E404D mutant is impaired, this does not
appear to stem from a defective binding of this substrate to the
transporter from the outside of the cell. We could investigate this by
taking advantage of the relatively high D-aspartate transport
catalyzed by the mutant. Unlabeled L-glutamate competes with
the D-[H]aspartate almost as effectively
as in the wild type (Fig. 7C). The same is true for
dihydrokainate, a nontransportable ring constrained analogue of L-glutamate (Fig. 7B). On the other hand, D-aspartate (Fig. 7A) as well as L-aspartate (data not shown) exhibit an affinity that is
apparently higher for E404D than for wild type transporters.
H]aspartate by Asp 398, Glu 404, and Asp 470
mutants. Hela cells were infected with recombinant vaccinia/T7 virus
and transfected with cDNA of the wild type (W.T), the
indicated mutants, or the vector alone. Results are given as
percentages of transport of the indicated mutant relative to the wild
type. Each bar is the mean ± S.E. of 3-7
different experiments. A, D-[
H]aspartate uptake. B, L-[
H]aspartate
uptake.
H]aspartate uptake by E404D mutant.
Hela cells were infected with recombinant vaccinia/T7 virus and
transfected with cDNA of the wild type or the E404D mutant. The
external medium contains the indicated concentration of the inhibitor. A, D-aspartate. B, dihydrokainate. C, L-glutamate. Results are given as percentages of
sodium-dependent transport of the indicated mutant relative to the wild
type. Each bar is the mean ± S.E. of 3 different
determinations.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.