From the Department of Neurobiology, University of Alabama at Birmingham, Birmingham, Alabama 35294-0021
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ABSTRACT |
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GABA1 transporters are
members of a large family of Na+-dependent
neurotransmitter reuptake proteins, located on the plasma membrane of
neurons and glia, that function in part to determine neurotransmitter
levels in the synaptic cleft (1). Demonstration of a physiological role
for GABA transporters comes from experiments involving specific GABA
uptake inhibitors; these inhibitors prolong the decay phase of
GABAA receptor-mediated post-synaptic potentials (2) and
both prolong the decay phase and increase the magnitude of responses
mediated by the G protein-coupled GABAB receptor (2-4).
GABA transporters also play a physiological role in toad and catfish
horizontal cells where calcium-independent GABA efflux through the
transporter is a principal mode of neurotransmitter release (5). GABA
transporters also have a pathophysiological role. There is decreased
calcium-independent GABA release in the affected hippocampus of
temporal lobe epileptics. This decrease in transporter-mediated efflux
is correlated with fewer GABA transporters and is hypothesized to
result in decreased inhibitory tone (6).
Not only can GABA transporters regulate neuronal signaling, transport
itself can be regulated. This is true for GABA transporters and other
members of this family as well (for review see Refs. 7 and 8).
Functional modulation occurs through a variety of second messengers
such as kinases, phosphatases, arachidonic acid, and pH. These factors
may act directly on the transporter protein (e.g. by
phosphorylation; see Refs. 9-12) or by regulating the interaction of
the transporter with other synaptic proteins, such as syntaxin (13). A
recurring theme is that the regulation occurs through changes in the
number of functional surface transporters (14-16).
The data are few regarding the physiological signals that trigger
functional transporter regulation. In rat basophilic leukemia cells,
the maximum velocity of serotonin transport is increased upon adenosine
receptor activation (17). Serotonin transport is increased in platelets
following stimulation of histamine receptors (18). Increases in
glutamate transport in primary astrocyte cultures are prevented by
antagonists of In the present report, we show that both agonists and antagonists of
the GABA transporter can trigger long term changes in GABA transporter
function, and we identify the mechanism underlying these changes. This
effect occurs in several different cell systems including hippocampal
cells that endogenously express the transporter. We show that
transporter expression increases with extracellular GABA concentration
and occurs on a time scale of minutes, consistent with the idea that
transporter function might be regulated in order to maintain constant
neurotransmitter levels at the synapse. Furthermore, we show that the
modulation occurs through the action of GABA on the transporter
directly (i.e. it is not mediated by GABA receptors) and
that the regulation of transporter expression levels is due to a net
change in the rate of transporter internalization.
Cell Culture--
Primary hippocampal cultures were prepared
from postnatal day 0-3 rats by mincing tissue in
1F9 cells (CHO cells stably expressing GAT1; see Ref. 23) were
maintained in [3H]GABA Uptake Assays--
Pre-assay drug
incubations were performed in HBSS. Preincubation solutions were
continually perfused to maintain constant extracellular drug
concentrations. Following preincubation, cells were rinsed three times
in 1× HBSS and allowed to equilibrate for 10 min in the final wash.
Buffer was then exchanged with control HBSS or drug-containing HBSS.
GABA was added to initiate the assay. The final [3H]GABA
concentration of the assay solution was 100 nM; the total GABA concentration of the assay solution was 30 µM. In
order to minimize changes in transporter expression during the assay,
assay times were 5 min. The assay was terminated by rapidly rinsing the
cells 3 times with 1× HBSS, followed by solubilization in 300 µl of
0.001-0.005% SDS at 37 °C for 2 h. Aliquots were used for
scintillation counting and to determine protein concentrations. Statistical analyses of the uptake data were performed using SPSS. Two-sample comparisons were made using t tests; multiple
comparisons were made using one-way analysis of variances followed by
Tukey's honestly significant difference post hoc test.
Biotinylation Experiments--
Biotinylation experiments were
performed essentially as described (14, 16). Cells were grown in 60-mm
tissue culture dishes to 80% confluence. The cells were rinsed twice
with 37 °C phosphate-buffered saline/Ca2+/Mg2+ (in mM: 138 NaCl,
2.7 KCl, 1.5 KH2PO4, 9.6 Na2HPO4, 1 MgCl2, 0.1 CaCl2, pH 7.4). The cells were next incubated with 2 ml of a solution containing 1 mg/ml sulfo-NHS biotin (Pierce) in
phosphate-buffered saline/Ca2+/Mg2+ for 20 min
at 4 °C with gentle shaking. The biotinylation solution was removed
by two washes in phosphate-buffered
saline/Ca2+/Mg2+ plus 100 mM
glycine and quenched in this solution by incubating the cells at
4 °C for 45 min with gentle shaking. The cells were lysed with 1 ml
of RIPA buffer (in mM: 100 Tris-Cl, pH 7.4, 150 NaCl, 1 mM EDTA, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 1 µg/ml leupeptin, 1 µg/ml aprotinin, 250 µM
phenylmethanesulfonyl fluoride) at 4 °C for 60 min. The cell lysates
were centrifuged at 20,000 × g at 4 °C for 60 min.
The supernatant fractions (300 ml) were incubated with an equal volume
of Immunopure Immobilized Monomeric Avidin beads (Pierce) at room
temperature for 60 min. The beads were washed three times with RIPA
buffer, and adsorbed proteins were eluted with SDS sample buffer (62.5 mM Tris-Cl, pH 6.8, 2% SDS, 100 mM
Western Analyses--
Analysis was performed on aliquots 1)
taken prior to incubation with beads (total cell lysate), 2) of the
supernatant fraction after adsorption and centrifugation (intracellular
fraction), and 3) of the bead eluate (biotinylated fraction). Western
blotting was carried out using anti-GAT1 antibody 346J (13) as
described (24) and visualized using ECL reagents (Amersham Pharmacia
Biotech). Monoclonal anti-actin antibodies (Sigma) were used to
normalize protein levels in each fraction. Immunoreactive bands were
scanned and quantitated with ImageQuant (Molecular Dynamics).
To examine the effect of extracellular GABA on chronic changes in
GABA uptake, primary neuronal cultures from neonatal rat hippocampus
were preincubated in 100 µM GABA for 1 h prior to assay. The results of this experiment are shown in Fig.
1A. Compared with control
cultures not incubated in GABA, treatment of these cultures with
extracellular GABA resulted in a greater than 2-fold increase in GABA
uptake measured in subsequent transport assays. This increase in
transport did not occur in experiments in which extracellular GABA was
replaced by glutamate or by glycine (data not shown). These data
suggest that the modulation of uptake is due to a GABA-mediated
process. The inclusion of SKF89976A, a high affinity inhibitor (25) of
the rat brain GABA transporter GAT1 (26), during the assay reduced GABA
uptake by greater than 90% both in control cultures and cultures
pretreated with GABA. These data suggest that the majority of GABA
transport mediated in these neuronal cultures, and the subsequent
modulation by GABA, occurs via GAT1. However, the effect is not
specific to GAT1 expressed in neuronal cultures. Fig. 1B
shows that a similar modulation in GABA uptake following extracellular
GABA treatment occurs in primary rat hippocampal astrocyte cultures.
Treatments with SKF89976A reveal that the effects on astrocyte cultures
are mediated primarily by GAT1 as well. To eliminate the possibility
that the increase in GABA transport following GABA preincubation might
simply be due to an increase in the velocity of transport (because of
higher GABA concentrations inside the pretreated cells), we performed the assays using 30 µM GABA, a concentration that is
saturating for GAT1-mediated transport.
-Aminobutyric acid (GABA) transporters on
neurons and glia at or near the synapse function to remove GABA from
the synaptic cleft. Recent evidence suggests that GABA transporter
function can be regulated, although the initial triggers for such
regulation are not known. One hypothesis is that transporter function
is modulated by extracellular GABA concentration, thus providing a
feedback mechanism for the control of neurotransmitter levels at the
synapse. To test this hypothesis, GABA uptake assays were performed on
primary dissociated rat hippocampal cultures that endogenously express
GABA transporters and on mammalian cells stably expressing the cloned
rat brain GABA transporter GAT1. In both experimental systems,
extracellular GABA induces chronic changes in GABA transport that occur
in a dose-dependent and time-dependent manner.
In addition to GABA, ACHC and nipecotic acid, both substrates of GAT1,
up-regulate transport; GAT1 transport inhibitors that are not
transporter substrates down-regulate transport. These changes occur in
the presence of blockers of both GABAA and
GABAB receptors, occur in the presence of protein synthesis
inhibitors, and are not influenced by intracellular GABA. Surface
biotinylation experiments reveal that the increase in transport is
correlated with an increase in surface transporter expression. This
increase in surface expression is due, at least in part, to a slowing
of GAT1 internalization in the presence of extracellular GABA. These data suggest that the GABA transporter fine-tunes its function in
response to extracellular GABA and would act to maintain a constant
level of neurotransmitter at the synaptic cleft.
INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
amino-3-hydroxy-5-methylisoxazole-4-propionic acid/kainate receptors (19); this result is consistent with the
hypothesis that extracellular transmitter levels, signaled by receptor
activation, feed back to up-regulate transporter function. Several
investigations have shown regulation of transporters following interactions of the transporter with transporter antagonists. Heterologously expressed norepinephrine transporters are down-regulated following long term (>3 days) treatment with the norepinephrine transporter antagonist desipramine, perhaps through changes in protein
expression and/or transporter turnover (20). For GABA transporters,
chronic treatment with the GAT1-specific transporter inhibitor
tiagabine down-regulates GABA transporter expression in brain tissue
(21), although whether this is due to inhibition of the transporter
directly or to spillover of GABA onto GABA receptors, and subsequent
receptor-mediated signaling effects, is not known.
EXPERIMENTAL PROCEDURES
Top
Abstract
Introduction
Procedures
Results
Discussion
References
-MEM supplemented
with cysteine, glucose, and 100 units of papain (Sigma or Worthington).
Tissue was incubated for 20 min at 37 °C followed by gentle
trituration, dilution, and plating onto
poly-L-lysine-coated glass coverslips. To obtain pure
neuronal cultures, mixed cultures were treated for 48 h with 10 µM cytosine arabinoside (Sigma); treatment was initiated
24 h after plating. Astrocyte cultures were prepared as described
(22). Cells were plated onto untreated 24-well plates and maintained in
Earle's MEM supplemented with 10% fetal bovine serum.
-MEM supplemented with 5% fetal bovine serum, L-glutamine, and penicillin/streptomycin. Transfections
were carried out using LipofectAMINE (Life Technologies, Inc.) in
Opti-MEM I (Life Technologies, Inc.). The lipid/DNA mixture was
incubated with the cells for 5 h; cells were then rinsed and
re-fed with complete media. Stable transformants were obtained by
selection in 500 ng/ml G418 (Life Technologies, Inc.).
-mercaptoethanol) at room temperature for 30 min.
RESULTS
Top
Abstract
Introduction
Procedures
Results
Discussion
References
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Fig. 1.
Extracellular GABA up-regulates GABA
transport in hippocampal cultures. A, up-regulation of
GABA transporter GAT1 by extracellular GABA. Drug concentrations (in
µM) are shown below the abscissa; values
underlined indicate that the drug was added only during the
assay. Primary neuronal rat brain hippocampal cultures were
preincubated for 1 h in control medium or medium containing GABA
or glutamate. Data are from four separate experiments, six
wells/condition/experiment. GABA uptake under control conditions ranged
from 432 to 765 fmol/min/mg of protein. Experimental conditions that
resulted in a significant change (p < 0.05) from
control values are denoted by the asterisk. B,
increases in GABA transport occur in both neurons and glia. Drug
incubation and symbols are as described in A. Data are from
three experiments, eight wells/condition/experiment. Mean neuronal GABA
uptake under control conditions was 624 fmol/min/mg of protein; mean
astrocyte GABA uptake under control conditions was 1123 fmol/min/mg of
protein. C, extracellular GABA concentration determines the
magnitude of up-regulation. Neuronal cultures were preincubated for
1 h in control medium or medium containing GABA at various
concentrations. Data are from two experiments, four
wells/concentration. Mean GABA uptake under control conditions was 372 fmol/min/mg of protein. D, GABA-mediated increases in
transport are time-dependent. Neuronal cultures were
treated with 100 µM GABA for various preincubation times.
Data are from two experiments, four wells/time point. Mean GABA uptake
under control conditions was 578 fmol/min/mg of protein.
Fig. 1, C and D, shows that the increase in transport following extracellular GABA treatment is both concentration-dependent and time-dependent. A logistic fit to the concentration-response data estimates an EC50 for up-regulation by GABA to be approximately 5 µM, which is comparable to the Km values for GABA uptake in both brain tissue (27, 28) and in cells heterologously expressing GAT1 (23, 26, 29). Additionally, maximal up-regulation of uptake occurred with 30 min of pretreatment with 100 µM GABA. Twelve-hour treatment with extracellular GABA resulted in no further increases in uptake than that seen at 30 min (data not shown).
The evidence that the EC50 of GABA necessary for up-regulation is comparable to the Km of the transporter for GABA suggested the hypothesis that the up-regulation was closely related to a transporter-mediated process. To add support to this hypothesis, experiments were performed (i) using other substrates and an antagonist of the transporter, and (ii) inhibitors of GABA receptors. The results of these experiments are shown in Fig. 2. Fig. 2A shows concentration-response curves for neuronal cultures treated with two GABA transport substrates, nipecotic acid and ACHC, and the GAT1 antagonist SKF89976A. Pretreatment of neurons for 1 h prior to assay with either nipecotic acid or ACHC resulted in an up-regulation in GABA transport, with estimates of EC50 values for up-regulation of approximately 9 and 72 µM, respectively. SKF89976A caused a down-regulation of transport, similar to that previously reported for the GAT1 antagonist tiagabine (21), with an EC50 for down-regulation of approximately 1 µM. To rule out the possibility that the decrease in transport seen following incubation with SKF89976A was due to acute inhibition of uptake during the assay (i.e. to rule out the possibility that SKF89976A was not washed off prior to assay), transport in untreated cells was compared with cells that were treated with SKF89976A for 5 min and then rinsed and assayed. No difference between these two groups was seen (data not shown). These results provide further evidence to support the hypothesis that the transport regulation is a transporter-mediated process. Namely, EC50 values for both up-regulation and down-regulation for each compound tested are comparable to the Km values for transport estimated for these compounds in previous GABA transporter investigations (30, 31).
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Because nipecotic acid, ACHC, and SKF89976A prevent the uptake of GABA competitively, it was possible that some of the transport regulation was due to GABA receptor-mediated action from neuronally released GABA. To test this hypothesis, GABA-mediated up-regulation was examined in the presence of bicuculline and phaclofen, inhibitors of GABAA and GABAB receptors, respectively. These results are shown in Fig. 2B. Pretreatment of cultures with these drugs, at concentrations that are routinely used to eliminate receptor-mediated responses in hippocampal cells, failed to inhibit the up-regulation. In addition, pretreatment of cultures with cycloheximide, a protein synthesis inhibitor, failed to alter the GABA-mediated up-regulation; these latter results are not surprising given the 30-min time course over which transport is altered (see Fig. 1D).
Given the data from hippocampal neurons suggesting that regulation of transport is mediated through GAT1, an expression system was sought that would mimic the endogenous phenomenology and that would permit a detailed characterization of the mechanisms underlying the regulation. Therefore, the experiments were repeated in 1F9 cells, a mammalian cell line stably expressing GAT1. The results of such experiments are shown in Fig. 3. As in neuronal cultures, pretreatment of 1F9 cells with 100 µM extracellular GABA resulted in a greater than 2-fold increase in subsequent GABA uptake. The other two transporter substrates, nipecotic acid and ACHC, similarly increased transport. The GAT1 antagonist SKF89976A, incubated alone or with submaximal concentrations of transporter substrates, reduced subsequent GABA transport. Similar results were obtained using a PC12 cell line stably expressing GAT1 (data not shown). These data demonstrate that GAT1 regulation occurs similarly in cells that endogenously express the transporter and in heterologous expression systems and support the idea that the regulation is a transporter-mediated effect. Given that there is unlikely to be extracellular GABA in these cultures in the absence of that exogenously applied, these data strongly suggest that the change in transport is not due to spillover of GABA onto GABA receptors.
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Chronic changes in uptake induced by transporter-interacting compounds could be produced, in general, either by altering the turnover rate of individual transporters or by altering the number of functional transporters. By analogy with receptor binding experiments, data obtained from saturation experiments are often used to distinguish between these two possibilities as follows: changes in the maximum velocity of transport (Vmax) are indicative of changes in the number of transporter-binding sites, and changes in affinity (Km) are indicative of changes in the function of individual transporters. Saturation analysis was performed on 1F9 cells preincubated in control solution or solution containing GABA or SKF89976A. The results of this experiment are shown in Fig. 4A. Eadie-Hofstee transformations (not shown) of the saturation data revealed Vmax values of 424 pmol/min/mg of protein (untreated cells), 302 pmol/min/mg of protein (SKF89976A-treated cells), and 842 pmol/min/mg of protein (GABA-treated cells). Km values, which were not significantly affected by the treatments were 5.3, 5.9, and 5.4 µM for untreated, SKF89976A-treated, and GABA-treated cells, respectively. These alterations in Vmax are consistent with changes in the number of functional transporters.
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To test this hypothesis directly, GAT1 immunoreactivity following biotinylation of surface proteins was examined in 1F9 cells preincubated with either GABA or SKF89976A. These data are shown in Fig. 4B. As shown both in the representative immunoblot and in the graph of densitometry measurements, preincubation of cells with GABA caused an increase in the amount of GAT1 immunoreactivity in the biotinylated fraction, the fraction corresponding to the surface population of transporters. This increase in surface immunoreactivity was correlated with a decrease in intracellular GAT1 immunoreactivity. Pretreatment of cells with SKF89976A resulted in a decrease in surface GAT1 immunoreactivity and a corresponding increase in intracellular GAT1 labeling. Two control experiments support these findings. First, the intracellular cytoskeletal protein actin was not labeled by the biotinylation reagent, suggesting that only surface proteins were being labeled; and second, immunoreactive bands were not seen in untransfected CHO cells immunoblotted with the GAT1 antibody (data not shown). The immunoblot data correlate well with the functional changes in uptake suggesting that compounds that interact with the transporter act to alter the number of cell-surface transporters. Furthermore, the evidence that the amount of transporter immunoreactivity in total cell lysates was unchanged by GABA or SKF89976A treatment supports the data showing regulation of the transporter in the presence of cycloheximide (see Fig. 2B) and suggests that the modulation is due to a redistribution of transporters rather than due to synthesis of new transporter protein.
The data obtained in the presence of GABA receptor blockers and the results from experiments using substrates of the transporter that do not activate GABA receptors strongly suggest that the mechanism of transporter regulation is not through a GABA receptor-mediated process. Another possibility is that the amount of GABA (or related compounds) present intracellularly regulates transporter redistribution. This hypothesis is consistent with up-regulation of GABA transport by transporter substrates and down-regulation of GABA transporters in the presence of SKF89976A. To test this hypothesis, 1F9 cells were transfected with another GABA transporter, GAT3, and experiments were performed in the presence of extracellular GABA and SKF89976A. If regulation is due to the amount of intracellular GABA, then GABA uptake by GAT3 in the presence of SKF89976A (which blocks GAT1 with approximately 200-fold higher affinity than GAT3) should increase GAT1 expression. On the other hand, if the regulation signal is a GAT1-mediated process directly, then the presence of SKF89976A should result in a down-regulation of GAT1 expression (as shown in Fig. 4B). The result of this experiment is shown in Fig. 5. The concentration of SKF89976A was chosen such that the majority of GAT1 would be inhibited, whereas GAT3 inhibition would be minimal. Cells expressing both GAT1 and GAT3 show less inhibition by SKF89976A than cells expressing GAT1 alone when SKF89976A is included in the assay. This decrease in inhibition of uptake is consistent with GABA transport occurring through GAT3. Biotinylation experiments (see immunoblot) revealed that surface GAT1 expression was reduced following preincubation with SKF89976A. Since intracellular GABA should have been accumulating (via GAT3) during this time, intracellular GABA levels do not appear to determine the regulation of the transporter. Rather, the down-regulation of the transporter in the presence of SKF89976A in these cells is consistent with an interaction of compounds with the transporter directly. It is interesting to note that preincubation of GAT1/GAT3-expressing cells with both GABA and SKF89976A caused a slight increase in transport compared with untreated cells. Since surface GAT1 expression was reduced by this treatment, such an increase is likely due to an increase in GAT3 expression.
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The redistribution of GAT1 from intracellular locations to the plasma membrane in the presence of extracellular GABA suggested that the modulation might be occurring by changes in the rates of transporter turnover. To test this hypothesis, 1F9 cells were preincubated for 1 h with a control solution or a solution containing 100 µM GABA, surface-biotinylated, and then processed at various time points after biotinylation. The results of this experiment are shown in Fig. 6A. Control cultures showed an approximately 50% decrease in surface GAT1 immunoreactivity (GAT1 immunoreactivity of the biotinylated fraction) over the 2-h experiment. In contrast, the decrease in surface GAT1 immunoreactivity was significantly slowed in GABA-treated cultures. Greater than 80% of the immunoreactivity remained at 2 h. Although these data strongly support the idea that the interaction of the transporter with substrates slows the internalization of the transporter, these data do not rule out the possibility that there is also an increase in the rate of transporter insertion into the membrane as well.
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To determine whether a similar mechanism occurs with the endogenous
GABA transporter, these time course biotinylation experiments were
repeated in hippocampal neurons. Cultures were preincubated for 1 h with a control solution or a solution containing 100 µM GABA, surface-biotinylated, and then processed at various time points
after biotinylation. The results of this experiment are shown in Fig.
6B. Once again, the evidence suggests that the interaction of the transporter with substrates slows transporter internalization.
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DISCUSSION |
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Neurotransmitter transporters exhibit a number of functional properties that enable them to influence extracellular neurotransmitter levels. For example, unitary transport rates are dependent on substrate concentrations, with half-maximal effective concentrations for transport occurring in the sub-micromolar to low micromolar range (1, 32). Additionally, transporters operate in reverse and non-vesicular efflux of transmitter will contribute to ambient extracellular transmitter levels. Transmitter efflux through the transporter may be related to pathophysiological conditions (6, 33, 34) but is also a principal mode of neurotransmitter release in some systems (5). Neurotransmitter transporter expression can be regulated, and such changes in the number of functional transporters will also contribute to the control of synaptic neurotransmitter levels. In the present report, we show that GABA transporter substrates and antagonists can interact with the GABA transporter to regulate directly transmitter uptake through alterations in surface transporter expression. These data strongly suggest a feedback mechanism in which transporters use extracellular neurotransmitter levels as a signal for the dynamic control of neurotransmitter levels at the synapse.
GABA transporter function is modulated by protein kinase C both in cells that endogenously express the transporter (13, 35) and in heterologous expression systems (24, 36, 37). The results from a majority of these studies show changes in the maximum velocity of transport, consistent with the hypothesis that transporter expression levels are altered. More direct measures, including subcellular fractionation (24) and estimating functional GABA transporter number by transporter-specific charge movements (15), support this hypothesis. One mechanism by which protein kinase C mediates its effect on GAT1 is by regulating the interaction of GAT1 with components of the docking and fusion apparatus (13). The evidence that GABA-mediated decreases in GAT1 function also result in a change in surface GAT1 molecules raises the possibility that such SNARE proteins are also involved in this form of GAT1 internalization. SNARE proteins have been implicated in the internalization and externalization of the type 4 glucose transporters (for review, see Ref. 38).
There appear to be multiple mechanisms even by which transporter substrates and/or antagonists can influence transporter function and expression. mRNA levels in brain for serotonin (39, 40) and dopamine (41) transporters are reduced following long term (>24 h) transporter antagonist treatments or by removal of substrate (42), although whether this significantly alters transporter protein levels (42) is unclear. In the present experiments it is unlikely that changes in mRNA or protein levels are mediating the regulation because (i) the time course of the modulation is on the order of minutes, (ii) it occurs in the presence of protein synthesis inhibitors, and (iii) GAT1 protein levels, although redistributed, appear to be unchanged. The triggers for these effects on mRNA levels are unclear since in vivo blockade of transporters likely causes increased receptor-mediated signaling as well.
Mammalian cells expressing norepinephrine transporters also show reduced uptake following 3-day transporter antagonist incubation, and this occurs in the absence of changes to mRNA levels (20). Whether this net internalization of norepinephrine transporters occurs by a mechanism similar to that of GAT1 in unknown; the much slower time course suggests that this could be a different form of transporter regulation. Experiments are under way to examine changes in GAT1 mRNA and protein levels following long term (>3 day) substrate and antagonist treatments. We are also examining whether other neurotransmitter transporters are regulated in a manner similar to GAT1. Although there have not been other reports of transporter regulation by extracellular neurotransmitter levels on the time scale of minutes, such changes may be easily overlooked because the time course of the regulation occurs on a time scale comparable to that typically used to assay uptake (i.e. 15-60 min). Thus, assays used to assess control levels of uptake will be confounded by concomitant changes in transporter expression.
There are two well characterized systems in which agonist-induced and
antagonist-induced signals produce changes in rates of internalization.
These are nicotinic acetylcholine receptors (43) and G protein-coupled
receptors (for review, see Ref. 44). Although the phenomenology of
these processes is well described, the mechanisms underlying the
down-regulation in surface expression are not well understood. How
rates of transporter internalization are altered by substrate
interaction is not known, although the opposite effects that occur in
the presence of antagonists raise the possibility that the
internalization signal is influenced by transporter conformation. One
possibility is that the process of substrate transport alters the
interaction of GABA transporter with SNARE proteins (e.g.
syntaxins) that act to sequester the transporter in a non-functional
state (13), thus shifting the balance of surface and internalized
transporter pools.
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FOOTNOTES |
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* This work was supported in part by the Epilepsy Foundation of America, W. M. Keck Foundation Grant 931360, and National Institutes of Health Grant DA10509.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Dept. of Neurobiology,
CIRC 446, University of Alabama at Birmingham, 1719 Sixth Ave. South,
Birmingham, AL 35294-0021. Tel.: 205-975-5098; Fax: 205-975-5097;
E-mail: quick{at}nrc.uab.edu.
The abbreviations used are:
GABA, -aminobutyric acid; ACHC, cis-1,3-aminocyclohexane
carboxylic acid; CHO, Chinese hamster ovary; HBSS, HEPES-buffered
saline solution;
-MEM,
-minimal essential media; RIPA, radioimmunoprecipitation assay.
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REFERENCES |
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