©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Conformational Changes Monitored on the Glutamate Transporter GLT-1 Indicate the Existence of Two Neurotransmitter-bound States (*)

Myriam Grunewald , Baruch Kanner (§)

From the (1)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

Membrane vesicles from rat brain have been subjected to trypsin treatment in the absence and presence of substrates of the (Na + K)-coupled L-glutamate transporter GLT-1. The fragments of this transporter have been detected upon immunoblotting employing several antibodies raised against sequences from this transporter. At the amino terminus, initially a fragment of an apparent molecular mass of 30 kDa is generated. This fragment is subsequently cleaved to one of 16 kDa. The generation of these bands is greatly inhibited in the presence of lithium. Moreover, lithium abolishes the positive cooperative activation of the transporter by sodium. The generation of the 30- and 16-kDa fragments is accelerated in the presence of L-glutamate and other transportable analogues, provided sodium is present as well. The 30-kDa fragment also contains an epitope from the loop connecting the putative membrane-spanning -helices 3 and 4. This epitope, in contrast with the amino-terminal one, is destroyed with time. The carboxyl-terminal epitope is predominantly located on a 43-kDa fragment which is slowly converted to one of 35 kDa. This conversion is not inhibited by lithium. It is, however, stimulated by L-glutamate and other transportable analogues, but only in sodium-containing media. Potassium also stimulates this conversion regardless of the presence of L-glutamate. The stimulation of generation of amino- and carboxyl-terminal fragments by L-glutamate is not mimicked by the non-transportable analogue dihydrokainate. However, the analogue blocks the stimulation exerted by L-glutamate. In addition to new experimental information on the transporters topology, our observations provide novel information on the function of the GLT-1 transporter. Although lithium by itself does not sustain transport, it may occupy one of the sodium sites and be transported. Furthermore, the transporter-glutamate complex appears to exist in at least two states. After the initial binding (suggested to be important for the decay of synaptic glutamate), it undergoes a conformational change which represents, or is tightly associated with, the transport step.


INTRODUCTION

Uptake of glutamate into nerve terminals and glial cells serves to keep its extracellular concentration below neurotoxic levels and helps in conjunction with diffusion to terminate its action in synaptic transmission (cf. Kanner and Schuldiner, 1987; Nicholls and Atwell, 1990; Mennerick and Zorumski, 1994; Tong and Jahr, 1994). The process is catalyzed by electrogenic sodium- and potassium-coupled L-glutamate transporters. Although not established definitely, the stoichiometry is likely to be two sodium ions accompanying each glutamate anion, while one potassium and one hydroxyl ion are transported out (Kanner and Sharon, 1978; Stallcup et al., 1979; Bouvier et al., 1992). 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 which 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 fine astroglial process of rat brain (Danbolt et al., 1992). The cloning and characterization of the human homologues of these proteins has recently been described (Arriza et al., 1994).

The relative role of diffusion and uptake of L-glutamate in the determination of its time course in the synaptic cleft is a subject of intensive investigation (Isaacson and Nicoll, 1993; Sarantis et al., 1993; Mennerick and Zorumski, 1994; Barbour et al., 1994; Tong and Jahr, 1994). The turnover number of the transporter has been estimated to be only a few per second (Danbolt et al., 1990). This is much too slow to account for the decay of L-glutamate in hippocampal synapses (Clements et al., 1992).

However, recent evidence indicates a possible role of glutamate reuptake in this process, and it was pointed out that the transporters could do so by binding the transmitter rapidly (Tong and Jahr, 1994). Binding of glutamate, a partial reaction of transport, could be much faster than the overall transport cycle. In order to probe the feasibility of this concept, we have investigated the conformational changes of the (Na + K)-coupled glutamate transporter GLT-1, expected to occur during its translocation cycle. Its susceptibility to trypsin, in the presence and absence of its substrates, was monitored using sequence-directed antibodies. Our data indicate that substrate binding causes changes in trypsin-sensitive sites throughout the protein and also provide the first experimental data on its topology. We provide evidence that there are at least two distinct transporter-glutamate complexes. In the first the position of glutamate can be occupied by the non-transportable analogue dihydrokainate. The transporter-glutamate complex, but not the transporter-dihydrokainate complex, can undergo a sodium-dependent conformational change which represents or is highly associated with the transport step.


EXPERIMENTAL PROCEDURES

Materials

Standard proteins for Tricine()-SDS-PAGE and Sephadex G-50 were obtained from Pharmacia LKB Biotechnol. Inc. Trypsin (diphenylcarbamyl chloride treated, from bovine pancreas, type XI), trypsin inhibitor (type I-S, from soybean), albumin bovine (fraction V), valinomycin, asolectin (soybean phospholipids, catalogue no. P.5038), cholic acid, and Tricine were purchased from Sigma. Nigericin was purchased from Calbiochem. Affi-Gel 15 and 10 were obtained from Bio-Rad. Nitrocellulose membranes (0.2 µm) were obtained from Hoefer Scientific Instruments. Tween 20 was purchased form J. T. Baker Inc. L-[H]Glutamate (60 Ci/mmol) was obtained from American Radiochemical Corporation and I-Protein A (130 µCi/ml) was obtained from DuPont NEN. All other reagents were analytical grade.

Asolectin was purified by acetone extraction (Kagawa and Racker, 1971), crude bovine brain lipids were extracted as described (Folch et al., 1957), and cholic acid was recrystallized from 70% ethanol (Kagawa and Racker, 1971) and neutralized with NaOH to pH 7.4. X-ray films were from AGFA.

Methods

Preparation of Crude Membrane Vesicles

Membrane vesicles from rat brain were prepared as described (Danbolt et al., 1990), except that the resuspension buffer contained 100 mM LiPi, pH 7.4, 5 mM Tris-SO, 1 mM MgSO, 0.5 mM Na-EDTA, and 1% glycerol.

Trypsin Digestion of Membrane Vesicles

Crude membrane vesicles (10-20 mg/ml) were rapidly thawed at 37 °C in a water bath, and after addition of 10 ml of either of the following loading buffer the incubation was continued for 10 more min. The composition of these buffers was 135 mM NaCl and 10 mM NaPi, pH 7.4; 135 mM KCl and 10 mM KPi, pH 7.4; 135 mM LiCl and 10 mM LiPi pH 7.4; or 135 mM Tris-HCl and 10 mM Tris-Pi, pH 7.4.

Then the vesicles were spun down by centrifugation at 4 °C for 15 min at 37,000 g, and the pellets were resuspended in the same solution at a protein concentration of 10 to 20 mg/ml. Subsequently, 30 µl of this suspension was diluted with 500 µl of the same solution with or without additions as detailed in the legends to the figures and incubated at 37 °C for 10 min. Unless indicated otherwise in the figure legends, L-glutamate was used at 1 mM. Then, 40 µl of a freshly prepared trypsin solution (dissolved in water) was added such that the protein ratio trypsin/membrane vesicles was 1:5. After the indicated times of incubation, the reaction was terminated by adding 600 µl of a solution of trypsin inhibitor (1 mg/ml). Immediately thereafter the mixture was diluted with 10 ml of ice-cold loading buffer containing 3% bovine serum albumin. Zero time points were performed by adding the trypsin inhibitor prior to trypsin, followed by immediate dilution in the ice-cold stop solution.

After centrifugation at 15,000 revolutions/min for 15 min, the pellets were washed with 10 ml of ice-cold sodium containing loading buffer and resuspended in the same buffer at a protein concentration of 3 mg/ml. The membrane vesicles were then processed for immunoblotting or transport as described below.

Assay of Trypsin Activity

This was performed as described (Reimerdes and Klostermayer, 1976).

Preparation and Purification of Antibodies

Antibodies were raised in rabbits against peptides corresponding to residues 17-32 (P-692, part of the amino terminus, RMHDSHLSSEEPKHRN), residues 151-167 (P-693, part of the putative external loop between transmembrane domains 3 and 4, KQLGPGKKNDEVSSLDA), to residues 496-512 (P-694, part of the carboxyl terminus, SKSELDTISDQHRMHED). The protocol described (Mabjeesh and Kanner, 1992) was followed.

The antisera were characterized against the HTP peak, a purified preparation of the L-glutamate transporter (Danbolt et al., 1990). They were affinity purified on Affi-Gel 10 coupled to P-692 or to Affi-Gel 15 coupled to either P-693 or P-694. The antibodies were tested against membrane vesicles from rat brain, using a 1:300 dilution. The 73 kDa was the only band detected by the anti-P-692 and the anti P-693 antibodies. In the case of the anti-P-694 antibodies, a minor 35 kDa band was also revealed in some preparations, probably representing a proteolytic fragment of the transporter.

The immunoreactivity of all the bands observed with the affinity purified antibodies was abolished by 50 nmol of the peptides used to raise them.

Reconstitution and L-Glutamate Transport

To membrane vesicles (120 µl, 3 mg/ml) were added (in this order) 15 µl of saturated ammonium sulfate; 168 µl of a 1:1 mixture of asolectin and brain lipids, 27 µmol total, suspended in 100 mM KPi, pH 7.4, 1% glycerol, 10 mM Tris SO pH 7.4, 0.5 mM NaEDTA, pH 7.4, 1 mM MgSO; 42 µl of NaCl 3 M and 22.5 µl of 20% cholic acid (neutralized with NaOH to pH 7.4. After incubation for 10 min on ice, the mixture was applied to three dried Sephadex minicolumns equilibrated with the above potassium-containing medium as described (Radian and Kanner, 1985). After they were collected by centrifugation, the proteoliposomes (20 µl) were assayed for (Na + K)-coupled L-[H]glutamate transport as described (Gordon and Kanner, 1988), except that millipore filters with 0.45-µm pore size were used.

After the assay, the proteins were quantified according to Peterson (1977). Glutamate transport in membrane vesicles (without reconstitution) was done as described (Kanner and Sharon, 1978).

Electrophoresis and Immunoblotting

Similar amounts of membrane suspensions (determined by the method of Bradford, 1976) were subjected to Tricine-SDS-PAGE and immunoblotted using the affinity purified antibodies, used at a 1:300 dilution, as described (Mabjeesh and Kanner, 1993). The standard proteins indicated in the figures are in kilodaltons.


RESULTS

Characterization of Peptide-specific Antibodies

Antibodies were raised against peptides corresponding to regions of the GLT-1 transporter predicted to be extramembranous (Pines et al., 1992) as illustrated in Fig. 1A. The peptides comprised residues 17-32 (part of the amino terminus, P-692), residues 151-167 (part of loop 3-4, the loop connecting putative transmembrane helices III and IV which contains the predicted N-glycosylation sites, P-693), and residues 496-512 (part of the carboxyl-terminal, P-694). In order to increase the probability that the antibodies raised against the individual peptides would also recognize the intact protein, the hydrophilicity and antigenicity profile of these peptides was determined according to the methods described (Hopp and Woods, 1981, 1983).


Figure 1: Location of chemically synthesized peptides within the GLT-1 sequence and specificity of antibodies generated against these peptides. A, regions of the sequence corresponding to synthetic peptides are outlined in black. The proposed model for the arrangement of the transporter in the membrane is taken from Pines et al. (1992). Potential tryptic cleavage sites (see ``Discussion'') expected to yield the 16-kDa (open triangles) and 30-kDa (arrowheads) amino-terminal fragments, as well as the 43-kDa (arrowheads) and 35-kDa (arrows) carboxyl-terminal fragments, are indicated. The potential glycosylation sites (Y) on the loop connecting helices III and IV are illustrated as well. B, specificity of the antipeptide antibodies. Equal amounts of membrane proteins (about 70 µg) were subjected to Tricine-SDS-PAGE and immunoblotted with affinity purified antibodies diluted at 1:300 in a solution of phosphate-buffered saline containing 1% bovine serum albumin and 0.002% azide. The antibodies used were raised against the highly purified transporter (lane 1), epitopes P-694 (lanes 2-4), P-693 (lanes 5-7), P-692 (lanes 8-10). 50 nmol of each of the corresponding homologous peptides were added to the solution containing the antibodies at their final dilution, prior to the incubation (lanes 3, 6, and 9). 50 nmol of the heterologous peptides were added in the same way using P-692 (lane 4), P-694 (lane 7), and P-693 (lane 10).



Indeed, with affinity purified antibodies raised against all three peptides, an intense band of around 73 kDa can be observed in the immunoblot of rat brain membranes (Fig. 1B, lanes 2, 5, and 8) which has a similar mobility as that observed with an antibody raised against the highly purified glutamate transporter (Fig. 1B, lane 1, Danbolt et al., 1992). The specificity of each of these antipeptide antibodies is illustrated by the ability of 50 nmol of the corresponding free peptide (homologous peptide) to inhibit the immunoreactivity (Fig. 1B, lanes 3, 6, and 9). Heterologous peptides do not inhibit (Fig. 1B, lanes 4, 7, and 10). Similar results are obtained using the HTP-peak fractions (Danbolt et al., 1990), a highly purified glutamate transporter preparation from rat brain (data not shown). The antibody raised against peptide P-694 was recently also shown to specifically react with the cloned and expressed glutamate transporter GLT-1 (Zhang et al., 1994).

Generation of Glutamate Transporter Fragments Containing the Amino Terminus

Membrane vesicles from rat brain have been incubated with trypsin for various times. After stopping the reaction with trypsin inhibitor and washing, the membrane proteins are separated on Tricine-SDS-PAGE and transferred to nitrocellulose. The fragments from the glutamate transporter which contain the amino terminus have been visualized with the anti-P-692 antibody (Fig. 2A). All bands visualized with this antibody, as well as with those described below, are specific. They were competed for by homologous but not by heterologous peptides (data not shown). In the presence of sodium ions, the band of 73 kDa corresponding to the intact transporter almost completely disappears, and bands of 30 and 16 kDa are generated. The kinetics suggest that the 30 kDa band emerges first, and subsequently the 16 kDa band is derived from it (Fig. 2A). The rate of conversion of the 30 to 16 kDa band is increased if in addition to sodium, glutamate is present as well (Fig. 2A). A pattern similar to that observed in the presence of sodium is also seen in the presence of potassium, except that now the effect of glutamate is smaller (Fig. 2A). The 30 kDa band is also generated in a lithium-containing medium, but its rate of conversion to the 16 kDa band is greatly slowed down whether glutamate is present or absent (Fig. 2A). Under these conditions the generation of the 30 kDa band from the full-length transporter is also retarded (Fig. 2A). In a control experiment we established that trypsin activity is not affected by sodium, potassium, and lithium ions and also not by glutamate (data not shown). The small effect of glutamate in potassium- and lithium-containing media is due to the fact that glutamate was added as its sodium salt, as it is not observed when the Tris salt of glutamate is used (data not shown).


Figure 2: Immunoreactivity and glutamate transport of the trypsin-treated membrane vesicles. A, membrane vesicles were loaded and treated with trypsin for the times (min) and conditions indicated (for further details, see ``Experimental Procedures''). About 40 µg of membrane protein were analyzed by Tricine-SDS-PAGE and immunoblotted with the anti-P692 antibody. B, the membrane vesicles were treated with trypsin under conditions identical to those in A (, lithium; , lithium + L-glutamate; , sodium; , sodium + L-glutamate). Subsequently, they were solubilized, reconstituted, and assayed for glutamate transport (2 min time points, ``Experimental Procedures''). Transport assays were done in duplicate; the size of the S.E. was smaller than the symbols.



We have also examined the functional consequences of the trypsin treatment on the transporter. We have used reconstitution to optimally express the transporters in the membrane vesicle preparation which were still active after the proteolysis. After stopping the reaction and extensive washing, the vesicles were solubilized with cholate and immediately reconstituted with a mixture of asolectin and brain lipids (Radian and Kanner, 1985) so that the internal medium contained potassium phosphate. The reconstituted proteoliposomes were diluted into the transport medium containing sodium chloride, L-[H]glutamate and the potassium-specific ionophore, valinomycin. These conditions (inwardly directed sodium and outwardly directed potassium gradients and an interior negative membrane potential) generate the maximal driving force for L-glutamate accumulation. It can be observed that the kinetics of inactivation of transport (Fig. 2B, a typical experiment, which was run in parallel to the one depicted in Fig. 2A) is only partly correlated to the conversion of the 30 to 16 kDa bands observed in Fig. 2A. Thus, the rate of inactivation when proteolysis is carried out in the presence of sodium is only slightly faster than when it is done in the presence of lithium (Fig. 2B). Interestingly, the rate of inactivation is increased in the presence of glutamate, but only when sodium is present (Fig. 2B). It is obvious that the correlation between the disappearance of the intact transporter (Fig. 2A) and the inactivation of transport activity (Fig. 2B) is partial. This discrepancy will be addressed under ``Discussion.''

The specificity of the effect of glutamate on the proteolysis of the 30 kDa amino-terminal fragment is shown in Fig. 3A. The conditions were chosen such that with sodium alone, both the 30 kDa as well as the 16 kDa bands are observed. Similar to the data shown in Fig. 2A, L-glutamate accelerates the proteolytic cleavage of the 30 kDa band (Fig. 3A, lane 3). The same effect is observed with L-aspartate (lane 7), D-aspartate (lane 8). All of these are substrates which are translocated by the glutamate transporter. On the other hand, the neurotransmitters GABA (lane 5), dopamine (lane 6), and serotonin (lane 9) did not exhibit the effect (Fig. 3A). The same result was obtained with D-glutamate (lane 4). This is in agreement with the well-known stereospecificity of the glutamate transporter (Balcar and Johnston, 1972). The effect of glutamate is dependent on its concentration. The half-maximal effect was observed at around 10 µM (Fig. 3B) which is somewhat higher than the observed K for transport (around 2 µM; Kanner and Sharon, 1978). However, under transport conditions, sodium outside, potassium inside (rather than sodium on both sides), the half-maximal effect of glutamate was shifted to lower concentrations, around 5 µM.


Figure 3: The effect of L-glutamate and other solutes on the generation of amino-terminal fragments of the GLT-1 transporter by trypsin. A, substrate specificity. Sodium-loaded membrane vesicles were digested for 5 min by trypsin in the presence of the following additions (1 mM): none (lane 2); L-glutamate (lane 3); D-glutamate (lane 4); GABA (lane 5); dopamine (lane 6); L- and D-aspartate (lanes 7 and 8, respectively), and serotonin (lane 9). The control, the intact transporter from membranes incubated without trypsin, is shown in lane 1. B, concentration dependence. Sodium-loaded membrane vesicles were digested as above in the presence of glutamate at the following concentrations: 0, 1, 10, 100, 1,000, and 10,000 µM (lanes 2-7). Control incubations without trypsin, with or without 10 mML-glutamate, are shown in lanes 8 and 1, respectively.



Generation of Glutamate Transporter Fragments Containing the Carboxyl Terminus

The data presented thus far can be explained by a sodium- and glutamate-dependent conformational change of the transporter, which causes two trypsin-sensitive sites, located in the amino-terminal half of the transporter, to become more exposed. The data presented in Fig. 4, in which an antibody against the carboxyl terminus was used to detect other fragments, indicate that this is also the case for at least one more site, located in the carboxyl-terminal part. In the presence of sodium alone trypsin treatment of the transporter results in the generation of a major 43 kDa band, as well as a minor band of 10 kDa. At longer times the minor 10 kDa band disappears, and part of the 43 kDa band seems to be converted into one of 35 kDa. In the presence of glutamate, the intensity of the 43 kDa band is reduced while the intensity of the 35 kDa band is increased. This phenomenon is a function of its concentration, just like that observed with the anti-P-692 antibody (data not shown). Also this effect appears to be a kinetic one as the phenomenon also occurs in the absence of glutamate but at longer times (data not shown). Again, the stimulation of the conversion of the 43 to 35 kDa band is observed with amino acid substrates of the transporter and not with those solutes which are not (data not shown). It should be pointed out that, unlike fragments containing the amino-terminal epitope P-692 (Fig. 2A and 3) or epitope P-693 (Fig. 8), those containing the carboxyl-terminal epitope P-694 give rise to a distinct pattern at the upper part of the gel (Fig. 4). This part contains a smeared background, probably reflecting fragments in different states of aggregation (Fig. 4). Although it is impossible to convey this feature, seen on the autoradiograph, to the photograph paper, it should be emphasized that superimposed on the background, the residual 73 kDa band corresponding to the intact transporter is present at strongly reduced levels.


Figure 4: Immunoreactivity of carboxyl-terminal tryptic fragments of the GLT-1 transporter. Membrane vesicles were loaded and treated with trypsin for the indicated times (min) and conditions. About 50 µg of membrane proteins were analyzed by Tricine-SDS-PAGE and immunoblotted with the P-694 antibody.




Figure 8: Immunoreactivity of the tryptic amino-terminal fragments with an antibody raised against the external N-glycosylated domain. The blot shown in Fig. 2A was stripped with 62.5 mM Tris-HCl, pH 6.7, 2% SDS, and 100 mM 2-mercaptoethanol for 45 min at 50 °C. After washing in phosphate-buffered saline containing 0.2% Tween-20, the blot was reprobed with the anti-P693 antibody.



As with the amino-terminal fragments, the ionic composition of the medium influences the trypsinization pattern, but in a slightly different way (Fig. 4). The conversion rate is increased by glutamate provided sodium is present. In the presence of lithium, a similar pattern is observed to the one with sodium, except for the lack of stimulation by L-glutamate (Fig. 4). On the other hand, in the presence of potassium, regardless of the presence of glutamate, the rate of conversion is similar to that observed in the presence of sodium and L-glutamate (Fig. 4).

Effect of Dihydrokainic Acid

Dihydrokainic acid is a competitive inhibitor of the GLT-1 transporter (Pines et al., 1992; Arriza et al., 1994) but appears not to be transported by it (Pocock et al., 1988; Barbour et al., 1991; Arriza et al., 1994). Thus, this compound may allow us to distinguish between the possibility that the effects of L-glutamate on the trypsinization pattern of the GLT-1 transporter are due to the mere binding of the substrate or one invoking a conformational change of the transporter, subsequent to the binding step. The experiment depicted in Fig. 5A, in which the anti-P-694 antibody is used, indicates that dihydroxykainate does not induce the sodium-dependent increase of conversion of the 43- to 35-kDa fragment. In this regard, it behaves like GABA, which is not a substrate, rather than like L-glutamate (Fig. 5A). However, while an excess of GABA does not affect the ability of L-glutamate to speed up the conversion of the 43 to the 35 kDa band, excess dihydroxykainate inhibits this action of L-glutamate (Fig. 5A). Similar results are obtained with the anti-P-692 antibody (Fig. 5B).


Figure 5: Effect of dihydrokainate on the generation of tryptic fragments of GLT-1. Sodium-loaded membrane vesicles were digested with trypsin for the indicated times and conditions. After Tricine-SDS-PAGE of the samples (around 50 µg of protein), they were immunoblotted with anti-P694 (A) or anti-P692 (B). The concentrations of the added compounds were: glutamate, 300 µM; dihydrokainate (DHK) and others (dopamine, DA) at 10 mM (A) or 5 mM (B).



Interaction of Lithium with the Glutamate Transporter

The conversion of the 30 kDa band to one of 16 kDa containing the P-692 epitope observed in the presence of sodium and potassium was greatly retarded when lithium containing media were used (Fig. 2A). Sodium and potassium are both substrates of the transporter. The retardation observed in the presence of lithium could be due to the fact that this ion is not translocated by the transporter. An alternative possibility is that lithium is not inert but is somehow interacting with the transporter and that this interaction is of a different nature than that with transportable ions. This was tested using substitution by Tris. It can be seen that with Tris present, the conversion of the 30 to the 16 kDa band is much more similar to that observed in a sodium or potassium medium than to that in lithium medium (Fig. 2A and 6). The conversion to the 16 kDa band was hardly influenced by L-glutamate, in contrast to the situation in sodium media. These observations suggest that the effects of lithium are due to a specific interaction of this cation with the transporter. Evidence for this at the functional level is seen in the experiment depicted in Fig. 7. The initial rate of glutamate transport depends on the sodium concentration in a sigmoid fashion when the ionic strength is maintained by Tris (Fig. 7). However, when lithium is used, the sodium dependence assumes the form of a rectangular hyperbola (Fig. 7). The small amount of uptake observed at zero-sodium may be due to the fact that glutamate (present at 10 µM) was added as its sodium salt. With choline substitution the sigmoid concentration dependence is observed as well (data not shown). The Hill coefficients calculated for this experiment are 1.01 for lithium and 2.00 for Tris substitution. The averaged (+S.E.) values are: lithium, 0.98 ± 0.03 (three experiments); Tris, 1.96 ± 0.06 (three experiments); and choline, 1.88 ± 0.02 (two experiments).


Figure 7: Effect of lithium on the sodium dependence of transport. Membrane vesicles were loaded with potassium and assayed for L-[H]glutamate transport in external media containing the indicated sodium concentrations. The total cation concentration was 150 mM and was achieved by supplementing the sodium chloride with chloride salts of lithium () or Tris (), or without addition (). Triplicate time points were taken (3 s, to ensure linearity) and 24 µg of protein was used per transport reaction. In each of the points the size of the symbols was larger than the S.E.



Characterization of the Amino-terminal Fragments of 30 and 16 kDa

The amino-terminal fragment of 30 kDa is expected to contain part of the large sugar-containing loop connecting helices III and IV (Fig. 1A), even if we take into account the faster mobility of hydrophobic proteins relative to hydrophilic protein standards (Tagaki, 1991). The amino-terminal fragment of 16 kDa most likely is generated by a cleavage of this loop very close to the extracellular end of helix III. This contention is supported by the experiment depicted in Fig. 8. This is exactly the same experiment from Fig. 2A in which the blot was stripped and reprobed with an antibody against the P-693 peptide (Fig. 1A). It can be seen that the kinetics of the generation of the 30-kDa fragment under the different ionic conditions are identical (Fig. 2A and 8). The 16-kDa fragment (Fig. 2A) is not detected by the anti-P-693 antibody. This indicates that the cleavage site generating this fragment is either closer to the amino-terminal than the P-693 epitope, or in the epitope itself. Tryptic cleavage at this site results in the loss of the P-693 epitope.


DISCUSSION

The data presented in this paper document that when membrane preparations from rat brain are digested with trypsin, the (Na + K)-coupled glutamate transporter is cleaved into several fragments (Fig. 2A, 4, and 8). The cosubstrate, potassium, increases the exposure of the trypsin-sensitive sites, leading to the generation of fragments containing the epitope P-694 located in the carboxyl terminus of the transporter (Fig. 4). It does not influence those cleavage sites involved in formation of the fragments containing the amino terminus (Fig. 2A). Cleavage at those latter sites however is greatly inhibited in the presence of lithium (Fig. 2A and 6). l-Glutamate and other transported amino acids enhance exposure of both types of cleavage sites in the presence of sodium (Fig. 2A, 3A, 6, and 8). These observations indicate that upon binding of its substrates, the transporter undergoes conformational changes, reflected in the altered accessibility of trypsin-sensitive sites.

The membrane vesicles containing the glutamate transporter GLT-1 appear to be predominantly of one orientation, since the P-693 epitope (Fig. 1A) disappears completely, at least in sodium and potassium containing media (Fig. 8). In a scrambled preparation, the epitope would have to be spared in the vesicles of the opposite orientation as the membrane would protect it against cleavage by trypsin. The possibility that the transporter resides mainly on unsealed membranes can be excluded, since the other epitopes tested (P-692 (Fig. 2A) and P-694 (Fig. 4)) are not destroyed by the protease. The minor 10 kDa band containing the P-694 epitope (Fig. 4) which is rapidly digested, could reflect a minor proportion of the vesicle population which is ``inside-out.''

The P-692 and P-693 epitopes are separated by a distance of only 119 amino acids (Fig. 1A). Therefore, we anticipated that the 30-kDa fragment containing the P-692 epitope located close to the amino terminus (Fig. 2A) also contains the P-693 epitope. In fact a band of this size lit up with the anti P-693 antibody (Fig. 8). Moreover, the kinetics of its appearance and disappearance under six different conditions was identical to that visualized by the anti-P-692 antibody (Fig. 2A and 8). This 30 kDa band is subsequently cleaved at least on an additional site to yield a 16 kDa band containing the P-692 epitope, which persists at the same steady state levels. On the other hand, the P-693 epitope disappears (Fig. 8). Therefore, those epitopes appear to reside on opposite sites of the membrane. The P-693 epitope is directed against the loop predicted to connect transmembrane helices III and IV which contains all the consensus sites for asparagine-linked glycosylation (Storck et al., 1992; Pines et al., 1992; Kanai and Hediger, 1992), and the glutamate transporters are in fact glycosylated (Danbolt et al., 1990; Danbolt et al., 1992; Storck et al., 1992). Since the P-693 epitope behaves like an external one, the membrane vesicles containing the glutamate transporter are predominantly right-side-out. This places the amino-terminal epitope P-692 on the inside, which is in harmony with the structural predictions (Storck et al., 1992; Pines et al., 1992; Kanai and Hediger, 1992). Implicit in these considerations is that the predicted topology is by and large correct from the amino terminus up to helix IV. In fact, the three groups independently predicted an identical topology up to helix VI (reviewed in Kanner, 1993). The hydropathy plot is less pronounced between the end of helix VI and the carboxyl terminus (Kanai et al., 1993). Our experimental system does not permit to determine the location of the cleavage sites. Right now we can only speculate on their location. The 30-kDa fragment containing both epitopes is probably generated by tryptic cleavage at lysine residues 226 and/or 230 and 231 located on the extracellular side of helix IV (Fig. 1A, arrowheads). This, together with the polysaccharides attached at asparagine residues 205 and 215, is expected to yield a glycoprotein band of a size close to that observed. Because of the atypical mobility of hydrophobic proteins (Tagaki, 1991), this conclusion is tentative at present. The secondary cleavage, yielding the 16 kDa band containing the P-692 epitope, is probably just extracellular of helix III at any of the lysine residues 148, 150, 151, 157, and 158 (Fig. 1A, triangles). Probably the cleavage is at least at 2 of these residues, as close inspection of Fig. 2A reveals that the 16-kDa band is in fact a doublet. The alternative positions for secondary cleavage, namely arginines 65 and/or 87, are highly unlikely as: 1) they are at either extreme of the loop connecting helices I and II and access of these sites to trypsin is highly unlikely, and 2) their size would be too short.

Both 43- and 35-kDa fragments containing the carboxyl-terminal P-694 epitope are preserved (Fig. 4) even up to 2 h of trypsin treatment (data not shown). Thus, it is likely that the epitope is located intracellularly, in agreement with the predictions (Kanner, 1993). One possibility is primary cleavage again at the lysines around position 230 (Fig. 1A, arrowheads) and the secondary sites lysine 302 or arginine 310 located in the loop connecting putative helices V and VI (Fig. 1A, arrows). This is supported by very recent experiments using an antibody raised against a peptide (P-695) comprising residues 372-386 of GLT-1. This epitope is located in the large putative intracellular loop connecting transmembrane helices 6 and 7 (see Fig. 1A). The same 43- and 35-kDa fragments were detected with antibody (data not shown). Thus, both primary and secondary cleavage sites must be located on the amino-terminal side of the P-695 epitope.

The cleavage site generating the 16 kDa from the 30-kDa fragment is more accessible in the presence of sodium ions as compared to lithium, and this effect is increased by glutamate (Fig. 2A). A small but consistent effect of sodium, with or without glutamate, was observed on the inactivation of glutamate transport activity by trypsin (Fig. 2B). However, it is clear that there is no direct relationship between this and the cleavage of a defined site (Fig. 2A). One possibility is that we are monitoring the cleavage of the GLT-1 transporter only, whereas other glutamate transporters (EAAC-I, GLAST) are present in the crude membrane vesicle preparation, which contribute to transport activity. This is unlikely, however, as the sequences of GLAST and EAAC-1 predict trypsin-sensitive sites in positions similar to where those of GLT-1 are thought to be. It appears more likely that the tryptic fragments of GLT-1 transporter, as long as they are residing together in the membrane, may still interact with each other and mediate glutamate transport. It is well known that assemblies of fragments of membrane proteins may preserve part or all of the functions of the intact protein (Liao et al., 1984; Karlish et al., 1990).

Comparison of Fig. 2A and 6 indicates that it is the presence of lithium which specifically reduces the accessibility of the cleavage site(s) yielding the 16-kDa fragment. A specific effect of lithium is also consistent with earlier findings indicating that not only trans-sodium but also trans-lithium is able to inhibit sodium-coupled L-glutamate fluxes (Pines and Kanner, 1990). Cations like choline and Tris, which presumably are inert, did not exhibit the effect. This led us to examine more closely the possible interaction of lithium with one or more of the sodium sites of the transporter. The results indicate that lithium, but not Tris (Fig. 7) and choline, abolishes the cooperativity of sodium binding to the transporter. A simple interpretation of this result is that of the two (or perhaps three) sodium sites, one is less specific so that lithium can occupy it and becomes translocated instead of one of the sodium ions. If no sodium is present then the other site(s) becomes perhaps also occupied by lithium, inducing a conformational change of the transporter not observed with other ions. This change is manifested only at the cleavage sites generating the 16- and 30-kDa amino-terminal fragments (Fig. 2A and 6) but not at that yielding the 35-kDa carboxyl-terminal fragment (Fig. 4). We propose that this represents a dead-end complex as no transport is observed in the sole presence of lithium (Fig. 7).

Glutamate and other transportable substrates have a relatively minor effect at the cleavage site generating the 16-kDa amino-terminal fragment (Fig. 2A, 3A, and 6) as compared to that yielding the 35-kDa carboxyl-terminal fragment (Fig. 4). The competitive analogue dihydrokainate which is not transported (Pocock et al., 1988; Barbour et al., 1991; Arriza et al., 1994) cannot mimic the glutamate effect at either of these two sites (Fig. 5, A and B). However, it can block the conformational changes induced by glutamate (Fig. 5, A and B). This indicates that there exist at least two forms of the transporter-glutamate complex. In the first form, dihydrokainate can bind to the glutamate site. This may represent the complex involved in the fast buffering glutamate at hippocampal synapses and thereby is helping to shape the time course of synaptic glutamate (Tong and Jahr, 1994). This probably would be most relevant to the analogous neuronal isoform of the transporter EAAC-1 (Kanai and Hediger, 1992; Rothstein et al., 1994). The glutamate-transporter complex but not the dihydrokainate-transporter complex undergoes the conformational changes documented in this paper. As these changes are sodium dependent, we propose that they represent or are tightly associated to the presumably much slower translocation step. This transport step in conjunction with diffusion, the relative contribution of the two being determined also by the geometry of the synapse (Barbour et al., 1994), is important to keep extracellular levels of glutamate below excitotoxic ones and of course affects synaptic transmission. The glial transporters GLT-1 (Pines et al., 1992) and GLAST (Storck et al., 1992) are suspected of playing a pivotal role in this.


FOOTNOTES

*
This work was supported by the Israel Science Foundation, 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; E-mail: KANNERB@HUJIMD.

The abbreviations used are: Tricine, N-tris(hydroxymethyl)methylglycine; PAGE, polyacrylamide gel electrophoresis; GABA, -aminobutyric acid.


ACKNOWLEDGEMENTS

We gratefully acknowledge the generous help of Drs. Gilia Pines and Nicola Mabjeesh in the first stages of this project. We also thank Yumin Zhang for his help with the preparation of the antibodies and Beryl Levene for expert secretarial help.


REFERENCES
  1. Arriza, J. L., Kavanaugh, M. P., Fairman, W. A., Wu, Y.-N., Murdoch, G. H., North, R. A., and Amara, S. G. (1993) J. Biol. Chem.268, 15329-15332 [Abstract/Free Full Text]
  2. Arriza, J. L., Fairman, W. A., Wadiche, J. I., Murdoch, G. H., Kavanaugh, M. P., and Amara, S. G. (1994) J. Neurosci.14, 5559-5569 [Abstract]
  3. Balcar, V. J., and Johnston, G. A. (1972) J. Neurobiol.3, 295-301 [Medline] [Order article via Infotrieve]
  4. Barbour, B., Brew, H., and Attwell, D. (1991) J. Physiol.436, 169-193 [Abstract]
  5. Barbour, B., Keller, B. U., Llano, I., and Marty, A. (1994) Neuron12, 1331-1343 [Medline] [Order article via Infotrieve]
  6. Bouvier, M., Szatkowski, M., Amato, A., and Attwell, D. (1992) Nature335, 433-435
  7. Bradford, M. M. (1976) Anal. Biochem.72, 248-254 [CrossRef][Medline] [Order article via Infotrieve]
  8. Clements, J. D., Lester, R. A. J., Tong, G., Jahr, C. E., and Westbrook, G. L. (1992) Science258, 1498-1501 [Medline] [Order article via Infotrieve]
  9. Danbolt, N. C., Pines, G., and Kanner, B. I. (1990) Biochemistry29, 6734-6740 [Medline] [Order article via Infotrieve]
  10. Danbolt, N. C., Storm-Mathisen, J., and Kanner, B. I. (1992) Neuroscience51, 295-310 [CrossRef][Medline] [Order article via Infotrieve]
  11. Folch, J., Lees, M., and Sloane Stanley, G. H. (1957) J. Biol. Chem.226, 497-509 [Free Full Text]
  12. Hopp, T. P., and Woods, K. R. (1981) Proc. Natl. Acad. Sci. U. S. A.78, 3824-3828 [Abstract]
  13. Hopp, T. P., and Woods, K. R. (1983) Mol. Immunol.20, 483-489 [CrossRef][Medline] [Order article via Infotrieve]
  14. Isaacson, J. S., and Nicoll, R. A. (1993) J. Neurophys.70, 2187-2191 [Abstract/Free Full Text]
  15. Jiang, J., Gu, B., Albright, L. M., and Nixon, B. T. (1989) J. Bacteriol.171, 5244-5253 [Medline] [Order article via Infotrieve]
  16. Kagawa, Y., and Racker, E. (1971) J. Biol. Chem.246, 5477-5487 [Abstract/Free Full Text]
  17. Kanai, Y., and Hediger, M. A. (1992) Nature360, 467-471 [CrossRef][Medline] [Order article via Infotrieve]
  18. Kanai, Y., Smith, C. P., and Hediger, M. A. (1993) Trends Neurochem. Sci.16, 365-370
  19. Kanner, B. I. (1993) FEBS Lett.325, 9599
  20. Kanner, B. I., and Schuldiner, S. (1987) CRC Crit. Rev. Biochem.22, 1-38 [Medline] [Order article via Infotrieve]
  21. Kanner, B. I., and Sharon, I. (1978) Biochemistry17, 3949-3953 [Medline] [Order article via Infotrieve]
  22. Karlish, S. J. D., Goldshleger, R., and Stein, W. D. (1990) Proc. Natl. Acad. Sci. U. S. A.87, 4566-4570 [Abstract]
  23. Liao, M.-J., Huang, K.-S., and Khorana, H. G. (1984) J. Biol. Chem.259, 4200-4204 [Abstract/Free Full Text]
  24. Mennerick, S., and Zorumski, C. F. (1994) Nature368, 59-62 [CrossRef][Medline] [Order article via Infotrieve]
  25. Nicholls, D. G., and Attwell, D. (1990) Trends Pharmacol. Sci.11, 462-468 [CrossRef][Medline] [Order article via Infotrieve]
  26. Peterson, G. L. (1977) Anal. Biochem.83, 346-356 [Medline] [Order article via Infotrieve]
  27. Pines, G., and Kanner, B. I. (1990) Biochemistry29, 11209-11214 [Medline] [Order article via Infotrieve]
  28. Pines, G., Danbolt, N. C., Bjoras, M., Zhang, Y., Bendahan, A., Eide, L., Koepsell, H., Storm-Mathisen, J., Seeberg, E., and Kanner, B. (1992) Nature360, 464-467 [CrossRef][Medline] [Order article via Infotrieve]
  29. Pocock, J. M., Murphie, H. M., and Nicholls, D. G. (1988) J. Neurochem.50, 745-751 [Medline] [Order article via Infotrieve]
  30. Radian, R., and Kanner, B. I. (1985) J. Biol. Chem.260, 11859-11865 [Abstract/Free Full Text]
  31. Reimerdes, E. H., and Klostermeyer, H. (1976) Methods Enzymol.45B, 26-28 [Medline] [Order article via Infotrieve]
  32. Rothstein, J. D., Martin, L., Levey, A. I., Dykes-Hoberg, M., Jin, L., Wu, D., Nash, N., and Kuncl, R. W. (1994) Neuron13, 713-725 [Medline] [Order article via Infotrieve]
  33. Sarantis, M., Ballerini, L., Miller, B., Silver, R. A., Edwards, M., and Attwell, D. (1993) Neuron11, 541-549 [Medline] [Order article via Infotrieve]
  34. Shafqat, S., Tamarappoo, B. K., Kilberg, M. S., Puranam, R. S., McNamara, J. D., Guadano-Ferraz, A., and Fremeau, R. T., Jr. (1993) J. Biol. Chem.268, 15351-15355 [Abstract/Free Full Text]
  35. Stallcup, W. B., Bulloch, K., and Baetge, E. E. (1979) J. Neurochem.32, 57-65 [Medline] [Order article via Infotrieve]
  36. Storck, T., Schulte, S., Hofmann, K., and Stoffel, W. (1992) Proc. Natl. Acad. Sci. U. S. A.89, 10955-10959 [Abstract]
  37. Tagaki, T. (1991) Adv. Electrophoresis4, 391-406
  38. Tolner, B., Poolman, B., Wallace, B., and Konings, W. (1992) J. Bacteriol.174, 2391-2393 [Abstract]
  39. Tong, G., and Jahr, C. E. (1994) Neuron13, 1195-1203 [Medline] [Order article via Infotrieve]
  40. Zhang, Y., Pines, G., and Kanner, B. I. (1994) J. Biol. Chem.269, 19573-19577 [Abstract/Free Full Text]

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