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INTRODUCTION |
Exocytosis takes place after an increase in the intracellular
calcium concentration and requires the fusion of synaptic vesicles, containing neurotransmitter, with the plasma membrane (1, 2). Three
membrane proteins are known to be involved in this process; synaptobrevin (VAMP),1
SNAP-25, and syntaxin 1 (3-5). SNAP-25 and syntaxin 1 form a complex
on the plasma membrane and function as a receptor for VAMP, present on
synaptic vesicles. When VAMP forms a heteromeric complex with syntaxin
1 and SNAP-25, synaptic vesicles come in close contact with the plasma
membrane and fusion between this membrane and the synaptic vesicle
membrane may occur (6).
Glycine is an inhibitory neurotransmitter mainly present in brainstem
and spinal cord, where it plays a role in a variety of motor and
sensory functions. Apart from its inhibitory effect on glycinergic
neurons, glycine modulates the action of glutamate on the
N-methyl-D-aspartate receptors. After the
release of glycine, high affinity reuptake mechanisms play an important
role in the removal of glycine from the extracellular space, thereby
terminating the neurotransmission signal. The uptake of glycine is
accomplished by two high affinity glycine transporters, GLYT1 and
GLYT2. Both transporters have been cloned (7-11) and have been the
subject of many studies (12). The fine tuning of reuptake mechanisms are thought to control the duration of the neurotransmission signal and
therefore the overall synaptic action (13).
Protein trafficking plays a key role in the regulation of many aspects
of neuronal function, and certainly many data point to the fact that
neurotransmitter transporters are also regulated by protein
redistribution mechanisms (14, 15). Recently we reported the
interaction between the glycine transporters and syntaxin 1 (16). This
interaction was found to be similar to those reported for the
-aminobutyric acid transporter GAT1 (17), the N- and Q-type calcium
channels (18), and the cystic fibrosis transmembrane regulator
(CFTR) chloride channel (19). All these studies have in common that
syntaxin 1 overexpression decreased the currents in the channels or the
transport rate in the transporters. The emerging view from these
studies is that syntaxin 1 somehow interferes in protein trafficking
(16, 20). The fact that syntaxin 1 is also involved in the fusion of
synaptic vesicles with the plasma membrane during the release of
neurotransmitter made us study the relation between exocytosis and
GLYT2 trafficking, both in the presence and absence of functional
syntaxin 1.
Here we show that a treatment that induces glycine release in
synaptosomes also stimulates the fast arrival of GLYT2 to the plasma
membrane, which is immediately followed by an internalization of the
transporter. The trafficking of GLYT2 toward and from the plasma
membrane might be supported by its localization in synaptic-like vesicles. We furthermore demonstrate that the arrival, but not the
retrieval, of GLYT2 from the plasma membrane is mediated by syntaxin 1.
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EXPERIMENTAL PROCEDURES |
Materials--
The Clostridium botulinum neurotoxins
Bont/C and Bont/B were purchased from Sigma. Bafilomycin A1 was from
Calbiochem (Bad Soden, Germany). Antibodies against syntaxin 1 (clone
SP6) and Na+/K+ ATPase (clone C464.6) were
obtained from Upstate Biotechnology (Lake Placid, NY).
Anti-synaptophysin (clone SP15) was from Calbiochem, and
anti-transferrin receptor was from Zymed Laboratories
Inc. (San Francisco, CA). GLYT2 antibodies have been
characterized (21).
Isolation of Synaptosomes and Synaptic
Vesicles--
Synaptosomes were purified from rat (3 months old, male)
brainstem and spinal cord as described (22), and resuspended in ice-cold HBM buffer (Hepes-buffered medium, composition in
mM: NaCl 140, KCl 5, MgCl2 1, Na2HPO4 1.2, NaHCO3 5, glucose 10, and HEPES-NaOH 20, pH 7.4). This buffer was used as a
Ca2+-free medium. The preparation was then incubated for 90 min at 37 °C with or without neurotoxin (80 nM Bont/C or
80 nM Bont/B) and immediately used in experiments.
Synaptic vesicles were isolated from rat (3 months old, male) brainstem
and spinal cord as described (23). Instead of the last purification
step (controlled pore glass chromatography), a flotation density
gradient was performed (24). In brief, crude synaptosomes (P2) were
prepared by differential centrifugation (10 min at 800 × g, followed by 15 min at 9200 × g on the
first supernatant) and lysed by a hypoosmotic shock. After differential centrifugation (20 min at 25,000 × g, supernatant was
taken and centrifuged for 2 h at 165,000 × g),
the obtained pellet was resuspended in 40 mM sucrose. This
preparation is referred to as crude synaptic vesicles (lysate of P2).
This fraction was then further purified by a continuous (50-800
mM) sucrose gradient. After centrifugation (3 h at 65,000 g), the 200-400 mM sucrose band was taken and 2 M sucrose was added to give a final concentration of 1 M. This sample was then transferred to a tube and subjected
to a flotation gradient by adding the following amounts of sucrose on
top of the sample: 6 ml of 800 mM, 6 ml of 600 mM, 6 ml of 380 mM, and 6 ml of 250 mM. After centrifugation (3 h at 27,000 × g), the band at the 600-380 mM sucrose
interface was taken, and the membranes isolated by centrifugation (2 h
at 165,000 × g). Finally, the pellet was resuspended
in 1 ml of glycine buffer (300 mM glycine, 5 mM
Hepes-NaOH, pH 7.3). During the purification, three samples were taken
for Western blot analysis corresponding to crude synaptosomes (P2),
crude synaptic vesicles (LP2), and purified synaptic vesicles (SV).
Release of [3H]Glycine from Synaptosomes--
A
suspension of synaptosomes (0.5 mg/ml of protein) in HBM buffer was
preloaded with 150 nM radiolabeled glycine (PerkinElmer Life Sciences) for 15 min. After centrifugation (3 min,
10,000 × g), the synaptosomes were washed 3 times with
HBM. Release of [3H]glycine was then evoked at 37 °C
by the addition of 1 mM 4-aminopyridine and 5 mM CaCl2 (final concentrations). After several
time intervals (0, 0.5, 1, 2, and 3 min), an aliquot (0.1 mg of
protein) was taken and immediately transferred to ice. Medium and
synaptosomes were then separated by centrifugation (3 min, 10,000 × g), followed by washing the synaptosomes three times with
ice-cold HBM buffer. Synaptosomes were finally resuspended in 200 µl
of 0.2 M NaOH. The [3H]glycine present in the
medium and in the resuspended synaptosomes was quantified by liquid
scintillation counting.
Uptake Assays--
Transport assays were performed at 37 °C
in HBM buffer as described (25) with some modifications. Synaptosomes
(0.5 mg/ml of protein) were incubated with 150 nM
[3H]glycine in a glass tube under continuous agitation.
Assays were performed in the presence of 1 mM sarcosine to
block glycine transport by GLYT1. After 0.5, 1, 2, and 3 min of
incubation, a sample (0.1 mg of protein) from the tube was taken and
immediately transferred to ice. After centrifugation (3 min,
10,000 × g), the synaptosomes were washed three times
with ice-cold HBM. Finally the synaptosomes were lysed in 0.2 M NaOH and the [3H]glycine present counted on
a liquid scintillation counter. In kinetic parameter determinations,
glycine concentrations ranging from 150 nM to 100 µM were used in a 1-min transport assay. All assays were
done in triplicate. Protein concentrations were determined by the
method of Bradford (26).
Biotinylation Studies--
At time point 0, 4-aminopyridine and
CaCl2 (final concentrations of 1 and 5 mM,
respectively) were added to a suspension of synaptosomes (0.5 mg/ml in
HBM). Immediately, at the indicated time points, aliquots (0.1 mg of
protein) were manually taken with a pipette and transferred to a tube
containing 1 ml of ice-cold HBM to stop all trafficking events.
Subsequently, samples were centrifuged (3 min, 10,000 × g), and the synaptosomes washed once with ice-cold HBM and
then resuspended in 1 ml of HBM containing the cell membrane
impermeable reagent
sulfo-N-succinimidyl-6-biotinamidohexanoate (2 mg/ml,
Pierce), and an incubation for 1 h at 4 °C was performed. Then,
the excess of reagent was quenched by adding lysine (100 mM
final concentration) and the incubation was continued for another 10 min. Samples were centrifuged (5 min, 10,000 × g), and
the pellets were washed twice with HBM and resuspended in lysis buffer (150 mM NaCl, 50 mM HEPES-Tris, 5 mM EDTA, 1% Triton X-100, 0.25% deoxycholate, 0.5% SDS,
0.4 mM phenylmethylsulfonyl fluoride, pH 7.4). The
biotinylated proteins were then incubated for 1 h with
streptavidin-agarose beads and precipitated by centrifugation (Sigma).
The beads were washed three times with 1 ml of lysis buffer, and the
proteins were eluted from the beads with Laemmli sample buffer and
subjected to Western blotting (25). Bands were visualized with the ECL
detection method (Amersham Pharmacia Biotech) and quantified on a model
300A densitometer in combination with ImageQuant software (Molecular
Dynamics, Sunnyvale, CA) by using film exposures that were in the
linear range. Standard errors in biotinylation studies were calculated
after densitometry from at least four separate experiments. To test the
biotinylation procedure, samples from different synaptosomal
preparations were taken and subjected to biotinylation.
Streptavidin-precipitated proteins were subsequently analyzed by
Western blotting with antibodies against intracellular proteins
(e.g. syntaxin 1 and synaptophysin). We never found
immunoreactivity against intracellular proteins in biotinylated
fractions, which indicated that only plasma membrane proteins were
biotinylated using the above described protocol.
Immunogold Labeling and Electron Microscopy--
Purified
synaptic vesicles were attached to collodion carbon-coated electron
microscopy grids, previously made hydrophilic by glow discharge. These
grids were placed on drops of a synaptic vesicle suspension (0.7 mg/ml
in glycine buffer; 300 mM glycine, 5 mM
Hepes-NaOH, pH 7.3) and incubated for 5 min at room temperature. After
removal of excess liquid, the absorbed material was fixed by incubation
with 0.5% glutaraldehyde in TBS (30 mM Tris, 150 mM NaCl, pH 8.2) for 2 min. Grids were then incubated for
10 min in TBG (TBS containing 0.1% bovine serum albumin and 1%
gelatin) to block nonspecific binding. Subsequently the grids were
transferred to drops of anti-synaptophysin (1/10 dilution) or
anti-GLYT2 (1/2 dilution) and incubated for 1 h. This step was
omitted in control incubations. Then, after several washes with TBG,
the grids were incubated for another 45 min with goat anti-mouse IgG
conjugated to 10-nm colloidal gold or 10-nm gold-protein A conjugate
for anti-synaptophysin or anti-GLYT2, respectively. Both secondary antibodies (Biocell Research Laboratory, Cardiff, United Kingdom) were
diluted 1/40 in TBG. Grids were then washed three times in TBS,
followed by three washes in glycine buffer, and fixed with 2%
glutaraldehyde in TBS for 2 min. Finally the grids were negatively stained for 40 s in a 2% aqueous solution of uranyl acetate.
Samples were examined in a JEM-1010 electron microscope, and pictures were taken using a 792 Bioscan digital camera (Gatan Inc., Warrendale, PA).
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RESULTS |
The starting point of the present study was to delineate the
physiological role of the interaction between syntaxin 1 and the
glycine transporter GLYT2. Since syntaxin 1 plays a crucial role in the
exocytosis of neurotransmitters and also interacts with several
neurotransmitter transporters, we decided to stimulate exocytosis in a
brain-derived preparation and study its effect on the glycine
transporter GLYT2, both in the presence and the absence of functional
syntaxin 1. The last is possible by inactivating syntaxin 1 in
vivo with Bont/C, a neurotoxin that specifically cleaves syntaxin
1 (27). For these studies purified sealed synaptic terminals
(synaptosomes) were used because these allow the treatment of Bont/C
and additionally contain the complete functional machinery of
exocytosis and reuptake of neurotransmitters.
Bont/C Inhibits Glycine Release from
Synaptosomes--
Synaptosomes were prepared from rat spinal cord and
brainstem because it has been reported that GLYT2, the model
transporter in our studies, is abundant in neurons from these areas
(28). Synaptosomes were preloaded with [3H]glycine,
and calcium-dependent exocytosis was provoked by plasma membrane depolarization using 4-aminopyridine in the presence of
extracellular Ca2+. 4-Aminopyridine blocks certain
voltage-gated K+ channels, causing spontaneous repetitive
firing in synaptosomes and a sustained rise in synaptosomal free
Ca2+ concentration (29, 30). Furthermore, it was suggested
that 4-aminopyridine closely mimicked physiological stimulation of calcium channels, resulting in a calcium influx (31). This treatment provoked a release of [3H]glycine from synaptosomes (Fig.
1), whereas no release was measured when
4-aminopyridine or extracellular calcium were added individually (data
not shown). A 30-min preincubation of the synaptosomes with 1.0 µM bafilomycin, an inhibitor of the V-ATPase that
prevents the entrance of neurotransmitter into synaptic vesicles (32), strongly reduced the release of [3H]glycine (Fig. 1).
This demonstrates that the release of [3H]glycine from
the synaptosomes is mainly due to exocytosis of [3H]glycine-filled synaptic vesicles. Accordingly,
preincubation of synaptosomes with Bont/C resulted in an inhibition of
[3H]glycine release, and a partial (approximately 55%)
proteolysis of syntaxin 1 (Fig. 1, inset). It has been
reported that Bont/C causes a 60% maximal breakdown of syntaxin 1 in
synaptosomes, and that this is sufficient to completely block vesicular
neurotransmitter release (27).

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Fig. 1.
Release of [3H]glycine from
purified synaptosomes. Synaptosomes were incubated for 90 min with
or without 80 nM Bont/C. During the last 15 min of
incubation, 150 nM [3H]glycine was added to
the synaptosomes. After washing, release from the synaptosomes was
evoked by adding 1 mM 4-aminopyridine and 5 mM
CaCl2, and the [3H]glycine released into the
medium was measured as described under "Experimental Procedures"
( , release without Bont/C (control); , release after Bont/C
incubation; , release after a 30-min preincubation with 1.0 µM bafilomycin). The inhibition in release after Bont/C
or bafilomycin treatment suggests that release is accomplished by
synaptic vesicles filled with [3H]glycine. Western blot
of synaptosomes (50 µg of protein/lane) shows the degradation of
syntaxin 1 by Bont/C, resulting in a product of approximately 30 kDa
(inset). Data from Bont/C and bafilomycin experiments are
significantly different from controls as determined by Student's
t test (p < 0.01, n = 4).
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Syntaxin 1 Inactivation Leads to a Reduction in GLYT2 on the
Plasma Membrane--
This study was performed on the glycine
transporter GLYT2, which, in contrast to isoform 1, is mainly
neuronal (28). Fig. 2A shows
the time course of [3H]glycine transport in synaptosomes
at a concentration of 150 nM [3H]glycine. The
uptake of [3H]glycine was completely abolished in the
absence of chloride ions in the uptake assay (Fig. 2A),
demonstrating that low affinity glycine transport systems, which do not
depend on Cl
ions (33), do not contribute to glycine
transport. Additionally, all transport assays were carried out in the
presence of sarcosine, a specific inhibitor of GLYT1. Therefore,
glycine transport in our experiments is mainly due to the activity of
GLYT2. A 90-min pre-treatment of synaptosomes with 80 nM
Bont/C reduced glycine transport by approximately 50% (Fig.
2A). A pre-treatment with Bont/B, a neurotoxin that blocks
neurotransmitter release by proteolytic cleavage of VAMP (34), reduced
glycine uptake in a similar way as Bont/C did (Fig. 2A).
Kinetic constants measurements showed that Bont/C treatment mainly
affected the Vmax of GLYT2
(Km = 26.7 ± 1.3 µM and
Vmax = 4.39 ± 0.18 µmol/mg of
protein/min for control synaptosomes, and Km = 20.1 ± 0.4 µM and Vmax = 2.27 ± 0.14 µmol/mg of protein/min for Bont/C-treated
synaptosomes). Changes in Vmax are usually
indicative for changes in the number of plasma resident transporters;
therefore, surface biotinylation experiments were performed to study
this issue in more detail. These studies revealed a reduction in
GLYT2-surface immunoreactivity after Bont/C (55% of control) or Bont/B
(65% of control) treatment (Fig. 2B), which explains the
observed inhibition in glycine uptake after neurotoxin treatment. This
is in agreement with that found for the
-aminobutyric acid
transporter GAT1 in hippocampal cultures, where GAT1 partially
disappeared from the plasma membrane after Bont/C incubation (35). Our
results show that two SNARE proteins (syntaxin 1 and VAMP) somehow
control the number of GLYT2 proteins on the plasma membrane. To
determine why GLYT2 disappears partly from the plasma membrane, we
hypothesized that neurotoxin treatment might interfere in GLYT2
trafficking and impede the transporter to reach the plasma membrane. To
study this issue, we analyzed in more detail surface expression of
GLYT2 in Bont/C-treated and non-treated synaptosomes.

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Fig. 2.
Uptake of [3H]glycine by GLYT2
in synaptosomes is inhibited by neurotoxin treatment.
A, , uptake without neurotoxin (control); , uptake
after Bont/C treatment; , uptake after Bont/B treatment; , uptake
in the absence of Cl ions. Transport assays were
initiated by the addition of 150 nM
[3H]glycine in the presence of 1 mM sarcosine
to inhibit transport by GLYT1. Data are from three separate experiments
and are significantly different from the control in a Student's
t test (p < 0.01). B,
synaptosomes were incubated with Bont/C (80 nM) or Bont/B
(80 nM) for 90 min, and the plasma membrane proteins
biotinylated. Biotinylated proteins were then isolated and subjected to
Western blotting using anti-GLYT2. These studies indicate that Bont/C
or Bont/B treatment reduce the number of GLYT2 proteins on the plasma
membrane as compared with a control without treatment. The
graph (inset in B) shows the densitometry
analysis of the bands obtained in the Western blots. Bars
represent the average of four independent experiments (mean ± S.E.). Asterisks indicate that results are significantly
different from control in a Student's t test
(p < 0.01).
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Syntaxin 1 Is Necessary in the Arrival of GLYT2 to the Plasma
Membrane--
As syntaxin 1 is both involved in exocytosis and in the
regulation of GLYT2, we provoked plasma membrane depolarization in synaptosomes by addition of 4-aminopyridine and extracellular calcium,
and studied its effect on GLYT2 trafficking by biotinylation. 4-Aminopyridine and CaCl2 were added to a suspension of
synaptosomes, and, at different time points, samples were taken and
placed on ice. Subsequently, the synaptosomes in these samples were
subjected to biotinylation and the amount of GLYT2 present on the
plasma membrane revealed by Western blotting with GLYT2 antibodies.
Fig. 3 shows that GLYT2 disappeared
gradually from the plasma membrane during conditions of sustained
exocytosis. This effect reached a maximum after 15 min and was not seen
when 4-aminopyridine or CaCl2 were individually added (data
not shown). Then, after 15 min of incubation, the remaining
extracellular calcium was removed by washing with EGTA, and the
synaptosomes were incubated for another 15 min in the absence of
4-aminopyridine and Ca2+. This treatment resulted in a
re-incorporation of GLYT2 into the plasma membrane of control
synaptosomes, although not completely to the level at the beginning of
the experiment. Even after longer incubation times, the starting level
could not be reached (Fig. 3). This may be explained by the fact that
EGTA only removes extracellular calcium and does not affect
intracellular levels. Anyhow, this experiment clearly shows that plasma
membrane depolarization in the presence of extracellular calcium
results in a removal of GLYT2 from the plasma membrane on a minute time
scale, and that removal of the extracellular calcium partly reverses
this effect. When this experiment was performed with synaptosomes
pretreated with Bont/C, GLYT2 was still able to leave the plasma
membrane after depolarization, but lost its capacity to return after
EGTA washing (Fig. 3). This last result demonstrates that syntaxin 1 is
necessary in the arrival and the incorporation of GLYT2 into the plasma
membrane, and is not involved in the removal of the transporter from
the plasma membrane. This may also explain why GLYT2 is less abundant
on the plasma membrane after Bont/C incubations (Fig. 2B).
During the 90-min preincubation with Bont/C, GLYT2 proteins are able to
leave the plasma membrane, but are subsequently unable to return,
resulting in an overall removal of GLYT2 from the plasma membrane after
the preincubation.

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Fig. 3.
Syntaxin 1 is necessary for the arrival to
but not for the removal of GLYT2 from the plasma membrane. At time
0, 1 mM 4-aminopyridine and 5 mM
CaCl2 (4AP/Ca2+) were added to
control synaptosomes. At the indicated time points, aliquots were taken
and immediately chilled on ice. After 15 min of incubation at 37 °C,
the remaining synaptosomes were washed with 2 mM EGTA to
remove all extracellular calcium, and, after another 15 and 30 min of
incubation, the final samples were collected. All samples were
subsequently subjected to biotinylation. The isolated biotinylated
proteins, which represent the plasma membrane fraction, were then
revealed by Western blotting using anti-GLYT2. The results show that
calcium stimulates the removal of GLYT2 from the plasma membrane on a
minute time scale, and after washing the transporter returns partly to
its origin (control). The same experiment was then performed with
Bont/C-treated synaptosomes. The results of this experiment are similar
compared with control synaptosomes, except that, after
removal of the extracellular calcium, GLYT2 is unable to return to the
plasma membrane. The graph shows the densitometry analysis
of the data obtained from the Western blots, and every bar
is the average of four independent experiments (mean ± S.E.).
Asterisks indicate that data are significantly different
from time 0 data as determined by Student's t test
(p < 0.01).
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Calcium Influx Results in an Immediate Incorporation of GLYT2 into
the Plasma Membrane--
To test if calcium also has a fast effect on
GLYT2 trafficking, we examined, in an experiment similar to that
described above, what happened to GLYT2 in the first few seconds after
plasma membrane depolarization and calcium influx. When 4-aminopyridine
and CaCl2 were added to synaptosomes at 37 °C, a slight
decrease in the amount of GLYT2 present on the plasma membrane was
immediately observed (data not shown). However, when the same
experiment was performed at 25 °C, an immediate up-regulation was
seen directly after stimulation (Fig. 4).
This effect was most prominent after the first second of incubation
with 4-aminopyridine and CaCl2, and was not detected when
4-aminopyridine and CaCl2 were individually added (data not
shown). The biotinylation method chosen to study this fast arrival of
GLYT2 to the plasma membrane allows to take samples as soon as 1 s, but it is not possible to study this phenomenon during the first
second. This is probably the reason why this effect was not observed at
37 °C because, at this temperature, the process is most likely much
faster and over when the first sample was taken. Nonetheless, with this
method, we were able to detect an immediate increase in the number of
GLYT2 on the plasma membrane by a factor of approximately 2.4 (2.4 ± 0.6; n = 4). This increase is immediately followed
by a retrieval of GLYT2 from the plasma membrane, as was already
demonstrated in Fig. 3. When the experiment was performed at 25 °C
with synaptosomes previously treated with Bont/C, this rapid increase
was not observed, again demonstrating that syntaxin 1 is necessary for
GLYT2 to arrive to the plasma membrane (Fig. 4).

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Fig. 4.
Fast effect of calcium on GLYT2.
Membrane depolarization in the presence of extracellular calcium
stimulates the incorporation of GLYT2 into the plasma membrane, which
is directly followed by its internalization. This effect was observed
by biotinylation studies performed as described in Fig. 3 at the
indicated time points and at 25 °C (control), and was abolished by
treating the synaptosomes with Bont/C. In the graph, the
up-regulation of GLYT2 is demonstrated after densitometry of the bands
obtained in the Western blots and shows the average of four independent
experiments (mean ± S.E.). Asterisk indicates that
data are significantly different from time 0 data as determined by
Student's t test (p < 0.05).
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GLYT2 Is Present in Synaptic-like Vesicles--
The active
trafficking of GLYT2 toward and from the plasma membrane prompted us to
investigate if GLYT2 is being transported by small vesicles. Therefore,
crude synaptosomes were lysed and the synaptic vesicle population
purified. During the purification, several samples were taken and
analyzed by Western blotting. Fig. 5A demonstrates the enrichment
of the synaptic vesicle marker synaptophysin in the purified fraction.
On the other hand, no plasma membrane contamination was present in the
purified synaptic vesicle preparation since no
Na+/K+ ATPase could be detected after the
purification. Immunoblots using an endosomal marker (transferrin
receptor) showed that fraction SV was almost free of endosomal
membranes. Syntaxin 1 was found to recycle with synaptic vesicles (36),
which explains why this SNARE protein was present in the purified
synaptic vesicle fraction. Interestingly, we detected GLYT2
immunoreactivity in the purified fraction (Fig. 5A),
although GLYT2 immunoreactivity was not enriched, suggesting that most
of GLYT2 is present on the plasma membrane or on other intracellular
compartments. Immunogold detection using anti-GLYT2 confirmed the
presence of GLYT2 in small (40-60-nm) synaptic-like vesicles (Fig.
5B). Approximately 5-10% of the vesicles were labeled with
anti-GLYT2, whereas synaptophysin-immunogold labeling on the same
vesicle preparation demonstrated that this synaptic vesicle marker was
present in most (80-90%) of the vesicles (Fig. 5C). These
experiments show for the first time GLYT2 immunoreactivity in small
synaptic-like vesicles, although the precise nature of these
GLYT2-containing vesicles has yet to be determined.

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Fig. 5.
Immunogold labeling on a synaptic vesicle
preparation demonstrates the presence of GLYT2 in this fraction.
Crude synaptosomes were purified from rat spinal cord and brainstem by
differential centrifugation (fraction P2) and subsequently lysed by an
osmotic shock. Then large membranes were removed by centrifugation (20 min at 25,000 × g) and the supernatant was subjected
to another centrifugation step (2 h at 165,000 × g) to
concentrate the remaining membranes. The pellet was resuspended
(fraction LP2) and layered on top of a continuous (50-800
mM) sucrose gradient. After centrifugation (3 h at
165,000 × g), the band (200-400 mM
sucrose) corresponding to synaptic vesicles was removed and used in a
flotation density gradient as described under "Experimental
Procedures." The band (380-600 mM sucrose) containing
the synaptic vesicles was subsequently isolated and called SV. The
amounts of synaptophysin (synaptic vesicle marker),
Na+/K+ ATPase (plasma membrane marker),
transferrin receptor (endosomal marker), syntaxin 1 (localized in
plasma membrane and synaptic vesicles), and GLYT2 in the samples taken
during purification (P2, LP2, and SV) were determined by Western
blotting (10 µg of protein/lane, A). The purified synaptic
vesicles were then immunogold labeled with anti-GLYT2 (B) or
anti-synaptophysin (C), negatively stained using uranyl
acetate, and examined by electron microscopy. Only small vesicular
membrane structures were seen in fraction SV. In most vesicles
(80-90%), anti-synaptophysin label was detected, whereas, in
approximately 5-10% of the vesicles, GLYT2 was labeled. In controls,
where the first antibody (anti-GLYT2) was omitted in the incubations,
no immunoreactivity was observed. Bar, 100 nm.
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DISCUSSION |
In the present study, we have shown that treatment of synaptosomes
with an agent that promotes plasma membrane depolarization in the
presence of extracellular calcium stimulates the arrival of the
neuronal glycine transporter GLYT2 to the plasma membrane, and that
this effect is immediately followed by an internalization of the
transporter. The arrival, but not the removal, of GLYT2 from the plasma
membrane is mediated by syntaxin 1 and probably other SNARE proteins.
Moreover, the present data demonstrate that GLYT2 is present in
synaptic-like vesicles, which might represent GLYT2 that is being
trafficked. These results reveal the close correlation between release
and uptake of neurotransmitter.
Regulation of GLYT2 by Calcium--
Recently the proline
transporter was found to be localized in synaptic vesicles (37), and
the noradrenaline transporter in secretory granules of PC12 cells (38).
Both neurotransmitter transporters belong to the same family as the
glycine transporters and are thought to be responsible for the
clearance of, respectively, proline and noradrenaline from the
extracellular space. Here data are presented that indicate GLYT2 is
also present in synaptic-like vesicles. At present, it is not known
whether transporters and neurotransmitters share the same vesicles or
use specialized individual vesicles. Nonetheless, a coordination in the
exocytosis of such vesicles should exist making a connection between
release of neurotransmitter and its reuptake by transporters. The
observation that the number of GLYT2 proteins on the plasma membrane
rapidly augments after membrane depolarization of nerve terminals now
strengthens this hypothesis of an exocytotic-like transportation of
neurotransmitter transporters from an intracellular compartment to the
plasma membrane. Jayanthi et al. (39) described a fast and
slow effect of calcium on the substrate-induced currents mediated by
the proline transporter. These experiments, however, were done in
transfected HEK cells, a cell line not known to have a specialized
exocytotic machinery. Additionally, previous work on the serotonin
transporter expressed in JAR cells also showed a long term inhibition
of calcium on the transport rate. This inhibition reached 85% after
1 h of incubation with the calmodulin antagonist CGS 9343B and was
found to affect mainly the Vmax of the serotonin
transporter (40). A decrease in Vmax is usually
indicative for a reduction in the number of transporters on the plasma
membrane. Taking all these data together, it seems that the long term
effect of calcium on the neurotransmitter transporters might be
modulated by protein kinases. This idea is strengthened by the fact
that activation of protein kinase C resulted in an inhibition of
glycine transport in glioblastoma cells (41); moreover, other
neurotransmitter transporters that belong to the same family as GLYT2,
like the serotonin and dopamine transporters and GAT1, were found to be
internalized upon protein kinase C mediation (42-44).
Fusion of synaptic vesicles with the plasma membrane, followed by the
compensatory endocytosis, induces the redistribution of certain
proteins present on the plasma membrane. This was recently found to be
the case for several SNARE proteins, e.g. syntaxin 1 and
SNAP-25 (45). These SNAREs, which are mainly present on the plasma
membrane, are internalized with vesicle proteins during endocytosis
coupled to exocytosis and are subsequently re-inserted into the plasma
membrane although at different rates (45). In addition, the dopamine
transporter was recently found to be internalized upon protein kinase C
activation and targeted to the endosomal/lysosomal pathway (43, 46).
This dynamin-dependent mechanism might also be used by
GLYT2 during coupled exocytosis/endocytosis. Future work, however,
should clarify the exact mechanism by which GLYT2 and possibly other
neurotransmitter transporters are being trafficked to and from the
plasma membrane upon calcium activation.
Physiological Role of Syntaxin 1 in Glycine Reuptake--
The
co-localization of GLYT2 and syntaxin 1 on the neuronal plasma membrane
and the functional and physical interaction between these two membrane
proteins suggested a physiological role for this interaction (16).
Overexpression of GLYT2 and syntaxin 1 in COS cells resulted in an
internalization of the transporter (16). However, the data presented
here obtained from a brain-derived preparation demonstrate that
syntaxin 1 does not stimulate internalization of the transporter but is
involved in its arrival to the plasma membrane. These seemingly
different results might be explained by a disruption in the equilibrium
of the SNARE system caused by overexpression of syntaxin 1, which may
vary the rate of exocytosis, protein trafficking, or even compete with
native SNARE proteins (20). Deken et al. (35) proposed both
a trafficking and a functional inhibitory role for syntaxin 1 in GAT1
regulation, and both processes might be separated in place and time. It
could well be that this also holds for GLYT2, although future
experiments may unravel the details of this possible dual role for
syntaxin 1. To conform to the results presented previously for GAT1,
where syntaxin 1 was proposed to be a positive regulator for GAT1
expression (35), we now propose that syntaxin 1 is involved in the
arrival and insertion of GLYT2 into the plasma membrane. These results demonstrated a syntaxin 1 involvement both in the fast arrival of GLYT2
after plasma membrane depolarization, as well as in calcium-independent arrival of GLYT2 to the plasma membrane. The last became evident from
the results presented in Fig. 3. After endocytosis of GLYT2 and
subsequent EGTA washing, GLYT2 was unable to return to the plasma
membrane in Bont/C-treated synaptosomes, in contrast to non-treated synaptosomes.
We have shown here that calcium controls the number of GLYT2 proteins
on the plasma membrane and that at least two SNARE proteins (syntaxin 1 and VAMP) are involved in GLYT2 trafficking. The results, therefore,
suggest that GLYT2 trafficking is accomplished by a process where
GLYT2-containing vesicles fuse with the plasma membrane. Previous
studies revealed a direct physical contact between syntaxin 1 and GLYT2
(16), similar to the one found for GAT1 (35). Moreover, these
interactions reduced transport rates in both transporters, suggesting a
regulatory inhibitory function for syntaxin 1. From the results
presented here, however, it does not become clear if GLYT2 trafficking
and its incorporation into the plasma membrane requires a direct
interaction between the transporter and syntaxin 1. However, it seems
reasonable to think that fusion of GLYT2-containing vesicles with the
plasma membrane, like synaptic vesicle fusion, requires only SNARE
proteins, and therefore the syntaxin 1-GLYT2 interaction might only
play a role in the inhibitory function of syntaxin 1.
In conclusion, calcium seems to have two effects on the arrival and the
retrieval of the glycine transporter GLYT2 toward and from the plasma
membrane. First, it causes a fast syntaxin 1-dependent
increase in the concentration of GLYT2 on the plasma membrane. This
up-regulation is directly followed by a removal of GLYT2 from the
plasma membrane under sustained exocytotic conditions. At the moment,
we can only speculate about the physiological role of these processes.
It could well be that the nerve terminal regulates in this way the
duration of the neurotransmission signal, both in the short term and
the long term. For the short term regulation of GLYT2, it seems
reasonable to think that during fast neurotransmission more GLYT2 might
be transiently needed to clear the neurotransmitter from the synaptic
cleft in an efficient way, keeping the neurotransmitter signal short.
On the other hand, the long term down-regulation of GLYT2 might result
in a longer presence of glycine in the synaptic cleft in situations of
sustained exocytosis. Within this context, it has been proposed that
the redistribution of membrane proteins may play an important role in
synaptic plasticity (15, 37). Without any doubt, many data now point to
the fact that the fine tuning of neurotransmitter transporters is
crucial in neurotransmission; as shown in this and other reports, this
mechanism should be controlled tightly in space and time.