From the
Membrane vesicles from rat brain have been subjected to trypsin
treatment in the absence and presence of substrates of the
(Na
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 (
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
Standard proteins for
Tricine
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.
Then the vesicles
were spun down by centrifugation at 4 °C for 15 min at 37,000
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.
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.
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).
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
The data presented in this paper document that when membrane
preparations from rat brain are digested with trypsin, the
(Na
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.
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.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
+ 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.
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).
+
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.
Materials
(
)-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.
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.
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.
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.
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.
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.
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.''
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.
+ 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.
-aminobutyric acid.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.