©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Glutamate 404 Is Involved in the Substrate Discrimination of GLT-1, a (Na K)-coupled Glutamate Transporter from Rat Brain (*)

(Received for publication, April 19, 1995)

Gilia Pines , Yumin Zhang , Baruch I. Kanner (§)

From the Department of Biochemistry, Hadassah Medical School, The Hebrew University, P. O. Box 12272, Jerusalem 91120, Israel

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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 + 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.


INTRODUCTION

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 + 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).

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 (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).

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 -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.


EXPERIMENTAL PROCEDURES

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.

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()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.

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.

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.


RESULTS

Fig. 1shows [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).


Figure 1: L-[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.




Figure 2: L-[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).


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 [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).


Figure 4: L-[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).


Figure 5: L-[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.


Figure 6: Uptake of the stereoisomers of [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.




Figure 7: Inhibition of D-[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.




DISCUSSION

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.


FOOTNOTES

*
This work was supported by the Israel Science Foundation, which is administered by the Israel Academy of Sciences and Humanities, the Charles H. Revson Foundation, and by the Bernard Katz Minerva Center for Cell Biophysics. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Fax: 972-2-757379; KANNERB{at}HUJIMD

The abbreviation used is: kb, kilobase.


ACKNOWLEDGEMENTS

We thank Dr. Bernard Moss for the provision of recombinant virus vTF7-3 and Beryl Levene for expert secretarial help.


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