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The Journal of Neuroscience, March 1, 2003, 23(5):1563
BRIEF COMMUNICATION
Plasma Membrane GABA Transporters Reside on Distinct
Vesicles and Undergo Rapid Regulated Recycling
Scott L.
Deken1,
Dan
Wang2, and
Michael W.
Quick2
1 Department of Neurobiology, University of Alabama at
Birmingham, Birmingham, Alabama 35294, and 2 Department of
Biological Sciences, University of Southern California, Los Angeles,
California 90089
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ABSTRACT |
Plasma membrane neurotransmitter transporters affect synaptic
signaling through transmitter sequestration. Transporters redistribute to and from the plasma membrane, suggesting a role for trafficking in
regulating synaptic transmitter levels. One method for controlling transmitter levels would be to regulate transporter redistribution in
parallel with transmitter release. Thus, how similar are these processes? We show that the trafficking of the GABA transporter GAT1
resembles the trafficking of neurotransmitter-filled synaptic vesicles:
(1) transporters located on the plasma membrane are internalized and
reinserted into the plasma membrane on the order of minutes; (2) the
rate of recycling is depolarization and calcium dependent; (3) GAT1
internalization is associated with clathrin and dynamin; and (4)
intracellular GAT1 is associated with multiple compartments and, more
importantly, is found on a distinct class of vesicles. These vesicles
are clear, ~50 nm in diameter, and contain many proteins found on
neurotransmitter-containing small synaptic vesicles; however, they
appear to lack several traditional small synaptic vesicle proteins,
such as synaptophysin and the vesicular GABA transporter. These data
provide additional support for the hypothesis that GABA transporters
traffic in parallel with neurotransmitter-containing small synaptic
vesicles and also raise the possibility that some fraction of vesicles
found in GABAergic neurons may not be participating in transmitter
release but rather in the rapid regulated redistribution of membrane
proteins involved in transmitter uptake.
Key words:
GAT1; neurotransmitter uptake; protein trafficking; recycling; synapse; synaptic vesicle
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Introduction |
At GABAergic synapses, removal of
neurotransmitter is mediated in part by the neuronal GABA transporter
GAT1. The importance of GAT1 is evidenced by the elongation of
postsynaptic currents and behavioral changes associated with its
inhibition (for review, see Beckman and Quick, 2000 ). Because transport
rates are relatively slow compared with the time course of
receptor-mediated signaling (Mager et al., 1993; Wadiche et al., 1995),
transporters are thought to exert their effects in part by acting as
diffusion sinks, buffering transmitter away from receptors (Tong and
Jahr, 1994; Wadiche et al., 1995). Therefore, altering the transporter
number at or near the synapse may play a role in regulating neuronal signaling.
Subcellular redistribution of transporters in neurons and glia is well
documented. The data for GAT1, and for some other members of this
family, are consistent with the hypothesis that neurons regulate GAT1
surface expression in parallel with extracellular transmitter levels.
One mechanism by which neurons could accomplish this task is to link
transmitter release with transmitter uptake. Thus, how similar are
transporter redistribution and synaptic vesicle recycling? The
transporters for GABA (Beckman et al., 1998), glycine (Geerlings et
al., 2000), and serotonin (Haase et al., 2001) interact with SNARE
(soluble N-ethylmaleimide-sensitive factor attached protein
receptor) proteins, including syntaxin 1A and Munc-18. This
interaction causes an increase in transporter surface expression (Deken
et al., 2000; Geerlings et al., 2001; Horton and Quick, 2001). The
dopamine transporter (DAT) is internalized via clathrin and sorted to
recycling or degradative pathways (Daniels and Amara, 1999; Melikian
and Buckley, 1999; Saunders et al., 2000); internalized DAT can
reappear on the plasma membrane, at least under particular
pharmacological manipulations (Carvelli et al., 2002). Furthermore,
electron microscopy experiments suggest that intracellular GABA
transporters (Barbaresi et al., 2001) and glycine transporters
(Geerlings et al., 2001) can be found on vesicles in presynaptic
terminals. Although these results suggest many similarities between
transporter trafficking and synaptic vesicle recycling, many questions
remain. What is the rate of transporter recycling? Is this rate
regulated by depolarization in a calcium-dependent manner? To which
intracellular compartments does GAT1 sort? Is the vesicle on which GAT1
resides the same or different from neurotransmitter-filled synaptic vesicles?
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Materials and Methods |
Cell culture and [3H]GABA
uptake assays. Hippocampal cultures were prepared from postnatal
day 0-3 rats (Beckman et al., 1999). Experiments were performed after
10-14 d in vitro. CHO cells were maintained in  -MEM
supplemented with 5% FBS, L-glutamine, and penicillin-streptomycin. Transfections were performed using
FuGene 6 (Roche, Indianapolis, IN) in Opti-MEM I
(Invitrogen, Rockville, MD). The lipid-DNA mixture
was incubated with the cells for 24 hr. Uptake assays were performed as
described previously (Bernstein and Quick, 1999). The final
[3H]GABA concentration was 40 nM; the total GABA concentration was 30 µM. Statistical analyses were performed using
SPSS (SPSS, Richboro, PA). Two-sample
comparisons were made using t tests; multiple comparisons
were made using one-way ANOVAs, followed by Tukey's honestly
significant difference post hoc test.
Immunocytochemistry and microscopy. Cells were plated onto
poly-L-lysine-coated coverslips and fixed with
4% paraformaldehyde, washed, incubated in blocking solution (10%
horse serum, 2% bovine serum albumin, and 0.25% Triton X-100 in PBS),
treated with anti-GAT1 (1:200) antibody, washed, treated with
biotinylated secondary antibody (1:250; Santa Cruz
Biotechnology, Santa Cruz, CA), and stained using Texas Red
(Vector Laboratories, Burlingame, CA). Cells were then
mounted, sealed with Vectashield (Vector Laboratories), and imaged with a laser-scanning confocal microscope (Olympus Fluoview;
Olympus Optical, Mellville, NY).
Biotinylation experiments. Biotinylation experiments were
performed as described previously (Whitworth and Quick, 2001). EZ-link Sulfo-NHS-SS-Biotin (Pierce, Rockford, IL) was used
to biotinylate cell surface proteins, and biotin stripping was
performed using 2-mercaptoethanesufonic acid.
Biochemical preparations. Synaptosomes were
prepared from rat cortices (Montague et al., 1994). Tissue was
homogenized in 0.32 M sucrose and centrifuged at
4200 × g for 2 min. The supernatant fraction was
centrifuged at 25,200 × g for 12 min. The resulting pellet was resuspended in 0.32 M sucrose and spun
at 25,200 × g for 12 min. This pellet was then
resuspended in HEPES-buffered saline solution (HBSS) and centrifuged at
11,000 × g for 5 min. Synaptosomes were fractionated
as described previously (Lim et al., 2001) by lysis in hypotonic media
on ice for 45 min and centrifuged at 2000 × g for 20 min. The resulting pellet was saved as the P1 fraction, and the
supernatant fraction was centrifuged at 32,800 × g for
1 hr. The resulting pellet was saved as the P2 fraction, and the
supernatant fraction was centrifuged at 100,000 × g
for 2 hr. The resulting pellet was saved as the P3 fraction, and the supernatant fraction was saved as the S3 fraction.
Flotation and immunoisolation. Flotation and immunoisolation
were performed as described previously (Zhai et al., 2001). Lysed synaptosomes were adjusted to 2 M sucrose and
loaded underneath a discontinuous sucrose gradient of 1.2, 1.0, 0.8, 0.6, and 0.3 M. The gradient was centrifuged at
350,000 × g for 3 hr. Protein A magnetic beads
(Dynabeads; Dynal, Great Neck, NY) were incubated overnight with a goat anti-rabbit or anti-mouse linker IgG
(Chemicon, Temecula, CA) at 10 µg/mg beads in borate
buffer. Beads were collected, washed with PBS-0.1% BSA, and blocked
(0.2 M Tris at pH 8.5 and 0.1% BSA) for 4 hr at
37°C. Linker IgG-coated beads were incubated overnight at 4°C with
rabbit polyclonal anti-GAT1 antibody or monoclonal anti-synaptophysin
antibody at a concentration of 10 µg/mg beads in incubation buffer
(PBS at pH 7.4, 2 mM EDTA, and 5% FBS). Primary
antibody-coated beads and control linker IgG-coated beads were
incubated overnight at 4°C with isolated fractions. Beads were collected, and the supernatant fractions were saved as nonbound fractions. The beads were washed five times with incubation buffer and three times with PBS for 10 min each and saved as bound fractions. Bound fractions were diluted to the same total volume as the
starting material. For electron microscopy, bead-bound fractions were
fixed in either 3% paraformaldehyde and 1% glutaraldehyde in 0.1 M phosphate buffer or 4% glutaraldehyde and
0.8% tannic acid in 0.1 M cacodylate buffer to
enhance the fixation. After fixation, the bead fractions were rinsed,
postfixed by 1% OsO4, dehydrated, and embedded (Zhai et al., 2001).
Antibodies were from the following sources: GAT1, vesicular GABA
transporter, synaptotagmin, and VAMP2 (vesicle-associated
membrane protein) were from Chemicon; synaptophysin
and syntaxin 1A were from Sigma (St. Louis, MO); rab3a was
from Santa Cruz Biotechnology; rab11 was from BD
Bioscience (Lexington KY); and SV2 was from
Calbiochem (San Diego, CA).
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Results |
After neurotransmitter release, synaptic vesicle proteins are
rapidly internalized via clathrin-mediated endocytosis (Heuser, 1989).
To determine whether GAT1 is internalized via this
mechanism, we used three approaches. The first approach
used hypertonic media to induce abnormal clathrin polymerization
(Heuser and Anderson, 1989). To control for the effect of sucrose on
exocytosis (Li et al., 2001), we first incubated dissociated
hippocampal neurons in high potassium for 15 min and then added various
concentrations of sucrose for 5 min. Treatment with 0.45 and 0.6 M sucrose caused an approximate twofold increase in
[3H]GABA uptake (Fig.
1A). This uptake was
blocked 94% by the GAT1-specific inhibitor SKF89976A (data not shown).
To determine whether this increase was attributable to increased
GAT1 surface levels, we performed surface biotinylation experiments.
Treatment of neurons with 0.45 M sucrose
increased GAT1 surface immunoreactivity, which correlated
with the functional increase (Fig. 1B). Because the multiple effects of hypertonicity on exocytosis and endocytosis complicate interpretation of these results, our second approach was to
determine whether GAT1 internalization was disrupted by expression of
an inactive form of dynamin, a protein that participates in
clathrin-mediated endocytosis (Damke et al., 1994). We overexpressed the dominant-negative dynamin construct K44A in CHO cells stably expressing GAT1 and examined GABA uptake and GAT1 surface expression. Compared with cells transfected with plasmid vector alone or with wild-type dynamin, cells transfected with K44A dynamin showed increased
GABA uptake (Fig. 1C) and increased GAT1 surface
immunoreactivity (Fig. 1D). The third approach was to
verify colocalization of GAT1 and a fluorescently labeled clathrin
construct in CHO cells (Fig. 1E). Together, these
data suggest that surface GAT1 is internalized via clathrin-mediated
endocytosis, consistent with the evidence that dynamin participates in
dopamine transporter internalization (Daniels and Amara, 1999; Saunders
et al., 2000).
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Figure 1.
GAT1 internalizes via clathrin. A,
GABA uptake in hippocampal neurons is increased in the presence of
sucrose. Cultures were incubated for 5 min in 90 mM
K+-HBSS and then incubated with the indicated
concentrations of sucrose in 90 mM
K+-HBSS for 5 min before assay. Data are from three
experiments, six wells per condition per experiment. Mean GABA uptake
under control conditions was 427 fmol/min per milligram of protein.
B, Hypertonic medium increases plasma membrane GAT1
expression. Surface biotinylation of GAT1 was performed in control
medium and 5 min after incubation in medium containing 0.45 M sucrose. Immunoblot shows GAT1 immunoreactivity in total
cell lysate (T), surface (S;
biotinylated), and internal (I; nonbiotinylated)
fractions. The graph quantifies GAT1 immunoreactivity in control and
0.45 M sucrose medium for surface (filled
bars) and internal (open bars) fractions. Data
are from three experiments. C, Expression of an inactive
dynamin construct increases GABA uptake. CHO cells were cotransfected
with vector alone (Mock), wild-type dynamin
(WT), or K44A dynamin (K44A)
constructs. Data are from two experiments, six wells per condition per
experiment. Mean GABA uptake under control conditions was 621 fmol/min
per milligram of protein. D, Expression of an inactive
dynamin construct increases plasma membrane GAT1 expression. Immunoblot
shows surface biotinylation of cells transfected as in
C. The graph quantifies results from three separate
experiments. E, GAT1 and a clathrin-green fluorescent
protein (Clathrin-GFP) construct colocalize in CHO
cells. *p < 0.05, conditions significantly
different from control.
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If GAT1 trafficking is similar to that of synaptic vesicle recycling,
then one would expect to find GAT1 associated with intracellular recycling compartments (such as endosomes) and vesicle fractions. We
used several approaches to test this hypothesis. The first approach
used sucrose gradient flotation assays (Fig.
2A). GAT1 associated
with organelles of buoyant density similar to the synaptic vesicle
protein synaptophysin (Takamori et al., 2000) but not to the
cytoplasmic protein Munc-18 (Rowe et al., 2001). To ensure that GAT1
and synaptophysin immunoreactivities were from membrane-bound organelles, we treated synaptosomes with Triton X-100 to disrupt membrane associations (Zhai et al., 2001). The shift to more dense sucrose fractions suggested that these proteins were no longer membrane-associated after detergent treatment.
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Figure 2.
Components of the GAT1 trafficking pathway.
A, Intracellular GAT1 has buoyant densities similar to
synaptophysin. Shown are immunoblots from a flotation assay of
synaptosomes stained with various antibodies. For the membrane
disruption experiments, synaptosomes were treated with 2% Triton X-100
for 45 min before flotation. B, GAT1 is found in
multiple cellular compartments based on isolation of purified organelle
fractions. C, GAT1 in nerve terminals is associated with
both endosomes and lysosomes. Immunoblots are from P2 fractions
immunoisolated with beads coated with GAT1 or irrelevant
(IgG) antibodies. NB, Nonbound fractions;
B, bound fractions. Data are representative of at least
three experiments.
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We next isolated purified organelles from hypotonically lysed
synaptosomes (Fig. 2B). Synaptophysin
immunoreactivity was enriched in the P3 fraction, consistent with the
idea that the P3 fraction represents the small vesicle fraction. rab5
immunoreactivity was found in the P1 and P2 fractions, supporting the
idea that these fractions are enriched in endosomes (Lim et al., 2001).
GAT1 was found in all pelleted fractions, consistent with its presence in multiple cellular compartments. We then immunoisolated GAT1 from the
P2 fraction. No immunoreactivity was detected in the bound fraction
using irrelevant antibodies. Immunoreactivity for the early and
recycling endosomal markers rab5 and rab11 and the lysosomal marker
lamp2 was detected in GAT1-bound fractions. Immunoreactivity for the
-subunit of the 20S proteosome was not detected in the GAT1-bound
fraction, suggesting that GAT1 is likely degraded by lysosomes and not
by proteosomes.
The association of GAT1 with the recycling endosome suggested the
hypothesis that the transporter is recycled back to the plasma membrane
after internalization. To test this hypothesis directly, we performed
reversible biotinylation experiments with cleavable biotin molecules.
Consistent with previous data (Whitworth and Quick, 2001), after 5 min
at 37°C, ~50% of surface biotinylated GAT1 in neurons was
internalized (Fig. 3A). When
the remaining cell surface biotin molecules were stripped and the
neurons were incubated at 37°C for 5 min, ~50% of the
internalized, surface-labeled GAT1 returned to the plasma membrane.
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Figure 3.
GAT1 recycling rates. A,
Surface-labeled GAT1 reappears on the plasma membrane. Surface GAT1
(S, left lane) was biotinylated and
placed at 37°C. After 5 min, the amount of internalized GAT1 was
determined (I). Surface biotin was then
removed, cells were placed at 37°C for 5 min, and surface GAT1
immunoreactivity was assessed after avidin precipitation
(S, right lane). B,
Surface biotin is completely removed. Surface GAT1 (S,
left lane) was biotinylated and reassessed after 5 min
at 4°C (S, middle lane). Surface biotin
was then removed, cells were placed at 4°C for 5 min, and surface
GAT1 immunoreactivity was assessed after avidin precipitation
(S, right lane). C, Avidin
added to intact cells only precipitates surface GAT1. Surface GAT1
(S, left lane) was biotinylated and
reassessed after biotin removal (S, second
lane from left). Avidin was then added to intact
cells or to lysed cells, and the resulting surface (S,
second lane from right) and intracellular
(I) GAT1 immunoreactivity was assessed.
D, GAT1 basal recycling reaches steady state in minutes.
Cells were treated as in A, except the second 37°C
step proceeded for the times shown above the immunoblot.
E, GAT1 recycling is regulated by activity in a
calcium-dependent manner. Experiments were performed as in
D for cultures untreated (filled
circles; 2 mM K+ and 2.5 mM Ca2+), treated with high
K+ (open circles; 90 mM
K+ and 2.5 mM Ca2+),
treated with high K+ in the presence of calcium
channel inhibitor (filled squares; 90 mM K+, 2.5 mM
Ca2+, and 200 µM
Cd2+), or treated with high K+ in
the absence of Ca2+ (open squares; 90 mM K+ and 1 mM EGTA).
Treatments began after 5 min internalization in control medium. Surface
GAT1 immunoreactivity, representing recycled GAT1, is plotted as a
percentage of GAT1 internalized in 5 min. Data are from four
experiments. Data in high K+ (open
circles) were significantly different from control at all time
points (p < 0.05).
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To be sure that we were only detecting GAT1 that had resurfaced, we
performed several control experiments. First, it was necessary to
ensure that the biotin that was being precipitated by avidin was
labeling resurfaced GAT1 only. That is, it was necessary to strip the
biotin from surface GAT1 that had not internalized. To verify this, we
repeated the experiments at 4°C to stop all protein trafficking, and,
as expected, GAT1 was not internalized from the plasma membrane (Fig.
3B, middle lane). These non-internalized plasma
membrane GAT1 proteins could not be precipitated with avidin after
biotin stripping, showing that the stripping procedure was efficiently
removing the biotin (Fig. 3B, right lane). We
next verified that the added avidin was only precipitating surface GAT1
molecules. We allowed biotinylated proteins to internalize for 5 min at
37°C and then stripped the remaining cell surface biotin at 4°C. We
were unable to precipitate GAT1 from the cell surface immediately after
biotin stripping (Fig. 3C, second lane from
left). We then incubated the neurons at 4°C for 5 min and were still unable to precipitate GAT1 from the plasma membrane (Fig.
3C, third lane from left). However,
when we permeabilized the cells, internal biotinylated GAT1 proteins
were readily precipitated (Fig. 3C, right lane).
These control experiments verified that we were precipitating only
biotinylated GAT1 that had returned to the cell surface.
We next used this method to determine the rate at which GAT1
recycles by permitting GAT1 internalization and then examining the
amount of GAT1 that returned to the surface after various lengths of
time (Fig. 3D). A time course for basal GAT1 recycling as a
percentage of GAT1 internalization after 5 min revealed that GAT1
recycling reaches steady state in ~5 min (Fig. 3E).
An important related question is whether the recycling of GAT1 is
regulated. To test this hypothesis, we determined the rates of GAT1
recycling in the presence of high extracellular
K+ concentrations. In the presence of 90 mM K+ (or in 30 mM
K+; data not shown), both the GAT1
recycling rate and the relative amount of GAT1 on the plasma membrane
increased compared with the basal state (Fig. 3E). This
increase in the presence of high K+ was
prevented by removing Ca2+ from the
extracellular medium (in the presence of EDTA) or using Cd2+ to block
Ca2+ channels. These data suggest that
GAT1 recycling is regulated in a
Ca2+-dependent manner.
The data in Figure 2B showed that GAT1
immunoreactivity was found in fractions enriched in vesicles,
consistent with electron microscopic data showing GAT1 on vesicles in
presynaptic terminals (Barbaresi et al., 2001). To determine the
molecular identity of these vesicles, we separated vesicles within the
P3 fraction by immunoisolation. We purified both GAT1-containing
vesicles and synaptophysin-containing vesicles with specific GAT1 and
synaptophysin antibodies conjugated to magnetic beads. Electron
microscopy revealed that GAT1-containing vesicles are clear synaptic
vesicles with a diameter of 47 ± 13 nm, similar to
synaptophysin-containing vesicles (Fig.
4A). The size of these
vesicles was normally distributed, suggesting the presence of GAT1 on a
homogenous population of vesicles.
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Figure 4.
The GAT1-containing vesicle. A,
GAT1 and synaptophysin are found on vesicles that are ~50 nm in
diameter. The P3 fraction was separated with GAT1 or irrelevant
(IgG) antibodies conjugated to beads and processed for
EM. B, The GAT1-containing vesicle is distinct from
neurotransmitter-filled synaptic vesicles. The bead-bound
(B) fraction and nonbound (NB)
supernatant fraction were immunoblotted with various antibodies to
proteins known to reside on synaptophysin
(SYPH)-containing vesicles. vGAT,
Vesicular GABA transporter.
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Does GAT1 reside on synaptophysin-positive synaptic vesicles? To test
this hypothesis, we examined proteins found on each of the
immunoisolated vesicle populations by immunoblot (Fig. 4B). Our data suggest that the GAT1-containing
vesicles are distinct from synaptophysin-containing vesicles. The
GAT1-containing vesicles appear to contain little or no synaptophysin;
synaptophysin-containing vesicles appear to contain no GAT1. In
contrast to synaptophysin-containing vesicles, the GAT1-containing
vesicles appear to lack SV2, synaptotagmin isoforms 1 and 2, and the
vesicular GABA transporter. However, the GAT1-containing vesicles and
synaptophysin-containing vesicles have in common syntaxin 1A, rab3a,
and synaptobrevin (VAMP2). GAT1-containing vesicles also contain rab11,
suggesting that these vesicles may be derived from the recycling
endosome (Ullrich et al., 1996).
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Discussion |
The mechanisms by which neurons regulate the release and uptake of
neurotransmitter is crucial for understanding synaptic signaling. The
present data provide direct evidence that the plasma membrane GABA
transporter GAT1 recycles to and from the plasma membrane in neurons
through a pathway similar to, and on a time scale comparable with, the
recycling of synaptic vesicles (Sudhof, 2000). The rate of recycling is
calcium dependent, suggesting that the triggers for transmitter release
are those that control transporter redistribution. The time scale over
which GAT1 recycles supports the hypothesis that neurons regulate
transporter expression and neurotransmitter release in parallel
(Beckman et al., 1998). One way that GAT1 expression and
neurotransmitter-filled synaptic vesicle fusion could be linked
mechanistically would be to have plasma membrane transporters expressed
on the same vesicles that contain neurotransmitter. However, although
we do find that GAT1 resides on a vesicle morphologically similar to
neurotransmitter-filled synaptic vesicles, these vesicles are not
classic small synaptic vesicles but rather vesicles that likely lack
synaptophysin and the vesicular GABA transporter.
Our data showing clathrin-mediated internalization and endosomal
sorting is consistent with that shown for DAT, suggesting that this is
a pathway shared by this family of plasma membrane transporters
(Daniels and Amara, 1999; Melikian and Buckley, 1999; Saunders et al.,
2000). We find GAT1 not only resides on endosomes in the nerve terminal
but also on a distinct class of synaptic vesicles. These vesicles could
represent populations that include endocytic, exocytic, or
Golgi-derived vesicles. GAT1 has been shown not to reside on
Golgi-derived vesicles containing the active zone assembly proteins
piccolo or bassoon (Zhai et al., 2001). These GAT1-positive vesicles
may be a cargo vesicle that assembles nonactive zone regions of the synapse.
Neurons control neurotransmitter levels in the synaptic cleft by
regulating release (Lin and Scheller, 2000). An emerging view is that
neurons can also control neurotransmitter levels by controlling uptake
rates (on a time scale of seconds) and transporter surface expression
(on a time scale of minutes) (Mennerick et al., 1999; Blakely and
Bauman, 2000; Deken et al., 2000; Robinson, 2002). Like
neurotransmitter-filled synaptic vesicle recycling, neurons may use
transporter recycling as a means of controlling these levels. The
present data support the hypothesis that GABA release is linked to its
subsequent reuptake. These paths may be similar such that the same
factors that regulate release can regulate reuptake. It may be that the
transporter resides on a distinct vesicle either because the
transporter needs to be recycled at regions distinct from the active
zone or because neurons need to fine tune transporter expression
independent of release.
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FOOTNOTES |
Received Oct. 31, 2002; revised Dec. 12, 2002; accepted Dec. 18, 2002.
This work was supported by National Institutes of Health Grants DA10509
and MH61468 (M.W.Q.). We thank S. L. Schmid for the wild-type and
K44A dynamin constructs and E. M. Lafer for the EGFP-clathrin
construct. We also thank R. G. Zhai, C. C. Garner, C. D. Gancayco, and W. J. Tyler for their expert assistance during the
course of this study.
Correspondence should be addressed to Michael W. Quick,
Department of Biological Sciences, HNB 228, University of Southern California, Los Angeles, CA 90089-2520. E-mail: mquick{at}usc.edu.
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TOP
ABSTRACT
Introduction
