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The Journal of Neuroscience, 1999:RC9:1-6
RAPID COMMUNICATION
Multiple G Protein-Coupled Receptors Initiate Protein Kinase C
Redistribution of GABA Transporters in Hippocampal Neurons
Matthew L.
Beckman1, 2,
Eve M.
Bernstein1, and
Michael W.
Quick1
1 Department of Neurobiology and 2 The
Medical Scientist Training Program, University of Alabama at
Birmingham, Birmingham, Alabama 35294-0021
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ABSTRACT |
Neurotransmitter transporters function in synaptic signaling in
part through the sequestration and removal of neurotransmitter from the
synaptic cleft. A recurring theme of transporters is that many can be
functionally regulated by protein kinase C (PKC); some of this
regulation occurs via a redistribution of the transporter protein
between the plasma membrane and the cytoplasm. The endogenous triggers
that lead to PKC-mediated transporter redistribution have not been
elucidated. G-protein-coupled receptors that activate PKC are likely
candidates to initiate transporter redistribution. We tested this
hypothesis by examining the rat brain GABA transporter GAT1
endogenously expressed in hippocampal neurons. Specific agonists of
G-protein-coupled acetylcholine, glutamate, and serotonin receptors downregulate GAT1 function. This functional inhibition is
dose-dependent, mimicked by PKC activators, and prevented by specific
receptor antagonists and PKC inhibitors. Surface biotinylation
experiments show that the receptor-mediated functional inhibition
correlates with a redistribution of GAT1 from the plasma membrane to
intracellular locations. These data demonstrate (1) that endogenous
GAT1 function can be regulated by PKC via subcellular redistribution,
and (2) that signaling via several different G-protein-coupled
receptors can mediate this effect. These results raise the possibility
that some effects of G-protein-mediated alterations in synaptic
signaling might occur through changes in the number of transporters
expressed on the plasma membrane and subsequent effects on synaptic
neurotransmitter levels.
Key words:
biotinylation; GAT1; hippocampus; neurotransmitter
uptake; protein trafficking; seven-helix receptor
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INTRODUCTION |
Plasma membrane GABA transporters
are members of a large family of Na+-dependent
neurotransmitter reuptake proteins, located on neurons and glia, that
function in part to regulate neurotransmitter levels in the synaptic
cleft. Not only can GABA transporters regulate neuronal signaling
(Dingledine and Korn, 1985 ; Hablitz and Lebeda, 1985; Solis and Nicoll,
1992; Isaacson et al., 1993), but transporter function also can be
regulated. Functional modulation, demonstrated for most members of the
transporter family (for review, see Gegelashvili and Schousboe, 1997;
Beckman and Quick, 1998), can occur through a variety of second
messengers. These factors can exert their effects directly (e.g., by
phosphorylation; Casado et al., 1993; Conradt and Stoffel, 1997; Huff
et al., 1997; Ramamoorthy et al., 1998) or by regulating the
interaction of the transporter with other nerve terminal proteins, such
as syntaxin (Beckman et al., 1998).
A common feature of many transporters is functional regulation by
protein kinase C (PKC) (Casado et al., 1993, Corey et al., 1994, Sato
et al., 1995; Loo et al., 1996; Conradt and Stoffel, 1997; Huff et al.,
1997; Qian et al., 1997; Apparsundaram et al., 1998b; Zhu et al.,
1998). The effects of PKC occur in part through changes in the
number of functional surface transporters (Qian et al., 1997; Quick et
al., 1997; Davis et al., 1998). The majority of experiments
demonstrating this effect have been performed on cloned transporters in
heterologous expression systems using pharmacological agents that
activate or inhibit PKC. Therefore, the physiological signals that
trigger PKC-mediated transporter modulation in endogenous systems are
not known.
Receptor-mediated signaling may be one such trigger. 5-HT transport can
be increased (1) by adenosine receptor activation in basophilic
leukemia cells (Miller and Hoffman, 1994) and (2) by histamine receptor
activation in platelets (Launay et al., 1994). In astrocytes, glutamate
transport is increased after glutamate application; the effect is
prevented with kainate receptor antagonists (Gegelashvili et al.,
1996). Signals mediated through G-protein-coupled receptors are a
likely trigger for PKC-mediated neurotransmitter transporter regulation
because (1) specific G-protein-mediated pathways result in PKC
activation, and (2) such receptors are abundant on both neurons and
glia. Support for this hypothesis comes from studies showing that the
norepinephrine transporter expressed in a neuroblastoma cell line can
be regulated by activation of muscarinic receptors (Apparsundaram et
al., 1998b). Unfortunately, expression levels of the transporter in
this system were too low to directly test whether modulation by
muscarinic receptors was correlated with a redistribution of the
transporter. In the present report, we show that several
G-protein-coupled receptors can regulate both GABA transporter function
and its subcellular distribution in neurons that endogenously express
these molecules.
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MATERIALS AND METHODS |
Cell culture. Primary hippocampal cultures were
prepared from postnatal day 0-3 rats by mincing tissue in  -MEM
supplemented with cysteine, glucose, and 100 U papain. Tissue was
incubated for 20 min at 37°C, followed by gentle trituration,
dilution, and plating. To obtain pure neuronal cultures, mixed cultures were treated for 48 hr with 10 µM cytosine arabinoside
(Sigma, St. Louis, MO); treatment was initiated 24 hr after plating.
[3H]GABA uptake assays. Neurons were
rinsed three times in 1× HBSS and allowed to equilibrate for 10 min in
the final wash. Drugs of interest were applied at the start of the
assay and remained present throughout (15 min). 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. The
assay was terminated by rapidly rinsing the cells three times with 1× HBSS, followed by solubilization in 300 µl of 0.005% SDS at 37°C for 2 hr. 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 ANOVAs
followed by Tukey's honestly significant difference post
hoc test.
Biotinylation experiments. Biotinylation experiments were
performed essentially as described (Qian et al., 1997; Davis et al.,
1998). Cells were grown in 100 mm tissue culture dishes to 80%
confluence. The cells were rinsed twice with 37°C PBS/Ca/Mg (in
mM: 138 NaCl, 2.7 KCl, 1.5 KH2PO4, 9.6 Na2HPO4, 1 MgCl2, and 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,
Rockford, IL) in PBS/Ca/Mg for 20 min at 4°C with gentle shaking.
Unless otherwise noted, drugs of interest were applied at room
temperature for 5 min before addition of biotin. The biotinylation
solution was removed by two washes in PBS/Ca/Mg 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
radioimmunoprecipitation assay (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, and 250 µM PMSF) 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 µl) 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, and 100 mM
 -mercaptoethanol) at room temperature for 30 min.
Western analyses. Analysis was performed on aliquots (1)
taken before 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 performed using anti-GAT1 antibody 346J (Beckman et al.,
1998) as described (Corey et al., 1994), and visualized using ECL
reagents (Amersham, Arlington Heights, IL). Monoclonal anti-actin
antibodies (Sigma) were used to normalize protein levels. Immunoreactive bands were scanned and quantitated with ImageQuant (Molecular Dynamics, Sunnyvale, CA).
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RESULTS |
To test the possibility that G-protein-coupled receptors are an
endogenous trigger for GABA transporter regulation, we applied the
G-protein-coupled acetylcholine receptor agonist muscarine to
hippocampal neurons and examined its effect on GABA uptake (Fig.
1A). Neurons treated
with a saturating concentration of muscarine showed an approximately
twofold decrease in GABA uptake. This decrease was mediated
predominantly by the rat brain GABA transporter GAT1, because
application of SKF89976A (Larsson et al., 1988), a GABA transporter
antagonist with relatively high affinity for GAT1 compared with other
GABA transporters, reduced GABA uptake in these cultures by >90%. The
muscarine-induced decrease in transport was prevented by co-application
of atropine, a muscarinic acetylcholine receptor antagonist. Nicotine,
a nicotinic acetylcholine receptor agonist with little effect on
muscarinic acetylcholine receptors, had no effect on GABA uptake. The
effect on GABA uptake was muscarine concentration-dependent (Fig.
1B), with a half-maximal effective concentration of
~820 nM. Taken together, these data suggest that
muscarinic acetylcholine receptors can mediate alterations in GAT1
function in hippocampal neurons.
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Figure 1.
G-protein-coupled receptors regulate GABA
transport in hippocampal cultures. A, Downregulation of
GABA transporter GAT1 function by muscarinic receptor activation. Drug
concentrations (micromolar) are shown below the
abscissa; drugs were present for the duration of the
assay. Data are from three experiments, four wells per condition per
experiment. GABA uptake under control conditions ranged from 574 to 813 fmol/min per mg of protein. *Experimental conditions that resulted in a
significant difference (p < 0.05) between
groups. B, Muscarine inhibition of GABA transport is
dose-dependent. Muscarine (filled circles) was
applied at the indicated concentrations. Addition of 10 µM atropine (open circles) was included in
some wells treated with 1 µM muscarine. Data are from
three experiments, three wells per condition per experiment. GABA
uptake under control conditions was 521 fmol/min per mg of protein.
C, Glutamate and serotonin also downregulate GABA
transport. Glutamate (open circles), serotonin
(filled circles), and dopamine
(filled square) were applied at the indicated
concentrations. Data are from two experiments, four wells per condition
per experiment. Mean GABA uptake under control conditions was 457 fmol/min per mg of protein. D, Multiple receptors likely
mediate decreases in GABA transport through the same mechanism. Drug
concentrations (micromolar) are shown below the
abscissa. Data are from two experiments, four to six
wells per condition per experiment. Mean GABA uptake under control
conditions was 761 fmol/min per mg of protein. *Experimental conditions
that resulted in a significant difference (p < 0.05) between groups.
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To see whether other receptors might also affect GABA transporter
function, we repeated the uptake studies in the presence of various
known transmitters that have receptors in the hippocampus (Fig.
1C). Both serotonin and glutamate induced decreases in GABA uptake in a dose-dependent manner; dopamine had no effect. These data
are consistent with the hypothesis that receptors that are coupled to
G-proteins that lead to PKC activation can regulate GABA transport.
Dopamine receptors, which couple to G-proteins that do not lead to PKC
activation, failed to have an effect.
Of course, muscarine, glutamate, and serotonin also activate receptors
other than those linked to increases in PKC. To determine specific
receptor classes that alter GABA uptake, we performed additional
experiments using specific receptor agonists and antagonists (Table
1). As also shown in Figure 1, the
nonspecific muscarinic acetylcholine receptor agonist muscarine
decreased GABA uptake. This decrease was blocked by atropine and
4-diphenylacetoxy-N-methyl-piperidine (4-DAMP), a muscarinic
acetylcholine receptor antagonist that has high affinity for the M1,
M3, and M5 muscarinic receptor subtypes. The decrease was not prevented
by himbacine, a receptor antagonist that has high affinity for the M2
and M4 receptor subtypes. These data support the idea that GABA uptake
can be regulated by the M1, M3, and M5 receptor subtypes; these
muscarinic subtypes specifically lead to PKC activation.
Glutamate also exerts its effects through G-protein-coupled receptors
(Table 1).
trans-(1S,3R)-1-amino-1,3-cyclopentanedicarboxylic acid (ACPD), a metabotropic glutamate receptor agonist,
decreased GAT1 function; this decrease was prevented by co-application
of methyl-4-carboxyphenylglycine (MCPG), a metabotropic receptor antagonist. Application of S-3,5-dihydroxyphenylglycine
(DHPG), a specific agonist of group I metabotropic receptors,
mimicked the trans-ACPD effects. L-AP-4, a
specific agonist of group III metabotropic receptors, did not affect
GABA uptake. Because group I metabotropic receptors are specifically
linked to PKC activation, glutamate also exerts its effects on GABA
uptake via receptors that activate PKC. The same is true of serotonin,
because -methyl(ME)-5-HT, a specific agonist of
PKC-activating 5-HT2 receptors, decreased GABA uptake.
One question is whether these multiple receptor systems can act
synergistically to regulate GAT1 function. To test this hypothesis, subsaturating concentrations of muscarine (0.3 µM) and
trans-ACPD (1 µM), applied individually and
together, were used to decrease GABA uptake (Fig.
1D). Although application of either agonist resulted
in a decrease in GABA transport, co-application further reduced GAT1
function. A related question is whether these different transmitter
systems are exerting their effects on the same population of
transporters. For example, the various receptors may be located on
different subpopulations of neurons. In this scenario, using saturating
agonist concentrations at each of two different receptors should lead
to a greater reduction in GABA uptake compared with the results
obtained stimulating either receptor alone. In the presence of both
muscarine (10 µM) and trans-ACPD (100 µM), the decrease in uptake was comparable with that seen
with either agonist alone. Taken together, these data suggest that
different G-protein-coupled receptors can act together to affect the
same population of GABA transporters.
Are the receptor-mediated decreases in GAT1 function PKC-dependent? To
answer this question, we examined muscarine-mediated changes in GABA
transport in the presence of activators and inhibitors of PKC (Fig.
2A). Consistent with
the idea that PKC and muscarine are acting through the same pathway,
application of the PKC-activating phorbol ester PMA mimicked the
muscarine-induced effect. Furthermore, co-application of both compounds
had no synergistic effect on transport. The inactive phorbol ester
4-phorbol 12,13-didecanoate had no effect on transport,
suggesting a PKC-mediated effect of PMA. Stronger support for the idea
that the muscarine-induced decrease in GABA transport is PKC-mediated
comes from experiments involving the PKC inhibitor staurosporine.
Co-application of staurosporine reversed both the PMA-induced decrease
and the muscarine-induced decrease in GAT1 function.
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Figure 2.
Receptor-mediated regulation of GABA transport
occurs via protein kinase C. Drug concentrations (micromolar) are shown
below the abscissa; drugs were present for the duration
of the assay. *Experimental conditions that resulted in a significant
difference (p < 0.05) between groups or
compared with control. A, Regulation of GABA transport
by muscarine in the presence of PKC activators and inhibitors. Data are
from three experiments, four to six wells per condition per experiment.
GABA uptake under control conditions ranged from 461 to 733 fmol/min
per mg of protein. B, Botulinum toxin prevents the
muscarine-mediated decrease in GABA transport. Data are from three
experiments, four to six wells per condition per experiment. GABA
uptake under control conditions ranged from 583 to 645 fmol/min per mg
of protein.
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Previously, we showed that PKC can mediate its reduction in GABA
transport, at least in part, by increasing the association of the
transporter with syntaxin 1A; specific cleavage of syntaxin 1A by
botulinum toxin C1 (BONT/C1) prevents the PKC-mediated decrease in GAT1
function (Beckman et al., 1998). Thus, if muscarinic receptor activation decreases GABA transport via PKC, cleavage of syntaxin 1A by
BONT/C1 should prevent the decrease. Application of BONT/C1 caused an
increase in GAT1 function (Fig. 2B); co-application of PMA or muscarine failed to alter this BONT/C1-mediated increase in
GABA transport, consistent with the idea that G-protein-coupled receptors can exert their effects on transport via PKC and syntaxin. Figure 2B also makes another important point.
Transport through GAT1 is voltage-dependent, and some of the decrease
in uptake seen with receptor activation could be attributable to
changes in membrane potential (e.g., via G-protein effects on ion
channels). The evidence that, in the presence of BONT/C1, uptake is
increased to similar values in the absence or presence of muscarine
argues against a decrease in uptake attributable to a decrease in
membrane potential. The evidence that PKC exerts its effects on
transport in cells under voltage-clamp conditions (Qian et al., 1997;
Quick et al., 1997) also argues against an effect attributable to
changes in membrane potential.
Previous studies in heterologous expression systems or in immortalized
cell lines showed that PKC can exert some of its effects by causing a
redistribution of transporters between the plasma membrane and the
cytoplasm. To test this hypothesis in primary hippocampal neurons
endogenously expressing the GABA transporter, and to provide further
support for the hypothesis that the receptor-activated effects on
transport are PKC-mediated, we performed saturation analyses on GABA
uptake and surface biotinylation experiments on GAT1 protein (Fig.
3). Untreated and muscarine-treated
neurons were subjected to uptake experiments at various GABA
concentrations. Saturation analysis demonstrated that muscarine induced
a reduction in the maximum velocity of transport, with no change to the
apparent affinity of GABA for the transporter (Fig. 3A).
These data are consistent with a reduction in the number of functional
transporters and comparable with previous data showing PMA modulation
of GABA transport (Beckman et al., 1998).
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Figure 3.
Receptor activation causes a redistribution of
GAT1. A, Saturation analysis of muscarine-mediated
changes in transport. GABA uptake in hippocampal neurons was determined
at various GABA concentrations in the absence (filled
circles) or presence (open circles) of 10 µM muscarine (left panel). An
Eadie-Hofstee transformation of these data is shown in the
right panel. Data shown are from one experiment, six
wells per concentration. The experiment was repeated twice with similar
results. B, Changes in surface GAT1 immunoreactivity as
assessed by surface biotinylation. Representative immunoblot shows GAT1
immunoreactivity from nonbiotinylated (I)
and biotinylated (S) fractions for cultures
untreated or treated with muscarine (10 µM) or PMA (1 µM). Quantitation of intracellular (open
bars) and surface (filled bars) GAT1
immunoreactivity is shown in the graph. Data, plotted as a percentage
of total immunoreactivity, are from densitometric measurements made on
three separate immunoblots. C, Time course of
muscarine-mediated GAT1 redistribution. Neurons were treated with
saline (untreated) or 10 µM muscarine for 1 min.
Biotinylating reagent was then added 0.5, 1, 2, or 5 min after
muscarine treatment. The representative immunoblots show GAT1
immunoreactivity for the biotinylated fraction at these time points.
Quantitation of GAT1 immunoreactivity is shown in the graph for neurons
in control solution (filled circles) or muscarine
(open circles).
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More direct evidence for a redistribution of transporters in response
to receptor activation comes from surface biotinylation experiments.
GAT1 immunoreactivity after biotinylation of surface proteins was
examined in untreated neurons and neurons treated with either muscarine
or PMA (Fig. 3B). As shown both in the representative immunoblot and in the bar graph of densitometry measurements (Fig. 3B, bottom panel), untreated neurons show a majority
of GAT1 immunoreactivity in the biotinylated (surface) fraction.
Incubation of cells with muscarine or PMA caused a decrease in surface
GAT1 immunoreactivity and an increase in the amount of GAT1
immunoreactivity in the nonbiotinylated (intracellular) fraction.
Comparable data were seen when neurons were treated with
trans-APCD (data not shown). The immunoblot data correlate
well with the functional changes in uptake, suggesting that
PKC-activating G-proteins may exert their effect on transport, at least
in part, by causing a redistribution of GABA transporters. The evidence
that PMA and muscarine produced comparable effects lends support to the
idea that muscarine mediates its effects through PKC. In addition, the
redistribution occurred in the presence of cycloheximide (data not
shown), suggesting that the modulation is attributable to a
redistribution of transporters rather than to synthesis of new
transporter protein.
The contribution of this form of transporter regulation to cell
signaling will in part be determined by the rate at which receptor-mediated redistribution of the transporter occurs. To determine this, we examined the amount of GAT1 immunoreactivity in the
biotinylated fraction of a cell treated with muscarine and then
biotinylated at 0.5, 1, 2, and 5 min after removal of muscarine (Fig.
3C). In untreated control cultures, the amount of GAT1
immunoreactivity in the biotinylated fraction remained constant over
the 5 min period; in the muscarine-treated cells, the amount of GAT1
immunoreactivity was reduced by 50% within 1 min of the end of
muscarine treatment. These results are consistent with the evidence
that muscarine reduces the number of surface GAT1 molecules and
demonstrate that the redistribution occurs rapidly after muscarine
treatment. The time course of the decrease in surface biotinylation in
muscarine-treated cells was comparable with the time course of uptake
inhibition measured in parallel cultures (data not shown).
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DISCUSSION |
G-proteins transduce signals from cell surface receptors for
neurotransmitters and other molecules to intracellular second messengers and ion channels (Simon et al., 1991; Hepler and Gilman, 1992; Conklin and Bourne, 1993). One subset of G-proteins (the Gq family) is responsible for coupling receptor-mediated
signals to phospholipase C activation; in turn, phospholipase C
catalyzes hydrolysis of phosphotidyl 4,5-bisphosphate to create
inositol trisphosphate and diacylglycerol. Inositol trisphosphate
causes release of Ca2+ from intracellular stores,
and diacylglycerol activates PKC (Berridge, 1993; Smrcka and Sternweis,
1993; Kuang et al., 1996). In the present report, we show that
activation of three different receptors that couple to this pathway can
reduce uptake of GABA in hippocampal neurons that endogenously express
these molecules. Furthermore, we show that the reduction in uptake is
PKC-mediated and is correlated with a redistribution of the GABA
transporter GAT1 from the plasma membrane to intracellular locations.
The evidence that botulinum toxin C1 prevents the receptor-mediated
inhibition of GABA uptake suggests that syntaxin 1A may play a role in
this process. We have shown previously that syntaxin 1A regulates GABA
uptake in heterologous expression systems and that this effect is
regulatable by PKC (Beckman et al., 1998). However, we do not have
evidence, to date, that syntaxin 1A also plays a role in the
subcellular redistribution of the transporter. The evidence that PKC
can regulate some transporters directly (Ramamoorthy et al., 1998)
suggests that multiple mechanisms may play a role in both transport
inhibition and transporter redistribution. Furthermore, we do not know
the extent to which net transporter internalization is responsible for
the decrease in uptake; our data only demonstrate that the time course
of these two events is similar after receptor activation.
Interestingly, maximal muscarinic receptor-mediated effects on GABA
transport and GAT1 transporter redistribution occurred within ~1 min.
This is faster than the muscarinic receptor effects on norepinephrine
transporters expressed in neuroblastoma cells (~30 min; Apparsundaram
et al., 1998a). Although this difference maybe attributable to
transporter differences in response to PKC, an intriguing possibility
is that in hippocampal neurons, the endogenous receptors, G-proteins,
protein kinases, and transporters form a tightly linked complex that
results in efficient transporter mobilization.
A physiological role for GABA transporters has been elucidated in
experiments involving specific GABA uptake inhibitors. These inhibitors
prolong responses mediated by GABAA receptors (Isaacson et
al., 1993), and both prolong and increase responses mediated by
G-protein-coupled GABAB receptors (Dingledine and Korn,
1985; Solis and Nicoll, 1992; Isaacson et al., 1993). GABA transporters also play a physiological role in retinal horizontal cells in which
efflux through the transporter is a principal mode of neurotransmitter release (Schwartz, 1987). Receptor-mediated modulation of
neurotransmitter transport may add to the repertoire by which
transmitter-mediated activity can regulate neuronal signaling. Because
receptor-mediated modulation results in a decrease in the number of
plasma membrane transporters, several mechanisms may play a role in
altering the neuronal signal. At slow synapses (see Lester et al.,
1994), the decrease in transmitter transport, per se, will alter
postsynaptic receptor-mediated responses. At fast synapses, signals
will be modulated because of a reduction in the number of diffusion
sinks (i.e., transporter binding sites) available for transmitter
sequestration (Diamond and Jahr, 1997). In addition, some transporters
exhibit both nonstoichiometric, substrate-dependent ionic fluxes and
substrate-independent leak currents (Sonders and Amara, 1996) that will
be reduced because of transporter redistribution.
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FOOTNOTES |
Received Jan. 6, 1999; revised March 10, 1999; accepted March 16, 1999.
This work was supported by W. M. Keck Foundation Grant 931360 and
National Institutes of Health Grant DA10509 (to M.W.Q.).
Correspondence should be addressed to Michael W. Quick, Department of
Neurobiology, CIRC 446, University of Alabama at Birmingham, 1719 Sixth
Avenue South, Birmingham, AL 35294-0021.
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