Download Dynamic Equilibrium of Neurotransmitter Transporters: Not Just for

J Neurophysiol 90: 1363–1374, 2003;
10.1152/jn.00317.2003.
review
Dynamic Equilibrium of Neurotransmitter Transporters:
Not Just for Reuptake Anymore
George B. Richerson and Yuanming Wu
Departments of Neurology and Cellular and Molecular Physiology, Yale University School of Medicine, New Haven 06520 and
Veteran’s Affairs Medical Center, West Haven, Connecticut 06516
Submitted 1 April 2003; accepted in final form 1 May 2003
Address for reprint requests: Correspondence and proofs to: George B. Richerson, MD, PhD, Neurology, LCI-704, Yale University School of Medicine, PO
208018, New Haven, CT 06520-8018 (E-mail: [email protected]).
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.
TRADITIONAL VIEW OF NEUROTRANSMITTER
TRANSPORTERS AS “VACUUM CLEANERS”
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Synaptic communication relies on rapid de-activation of
postsynaptic receptors to terminate synaptic responses. In
many synapses, specific neurotransmitter transporters on neurons and glia contribute to this de-activation by clearing neurotransmitter from the synaptic cleft. These neurotransmitter
transporters are members of a large superfamily with four
major branches: Na⫹- and Cl⫺-dependent symporters (e.g., for
GABA, monoamines and glycine), Na⫹- and K⫹-dependent
antiporters (e.g., for glutamate), vesicular transporters, and
amino acid transport systems. The Na⫹- and Cl⫺-dependent
symporters can be divided into four subfamilies in mammals
(Fig. 1A). In each case, these transporters utilize the energy
available from the transmembrane ion gradients and membrane
potential to provide the energy to drive their neurotransmitter
substrate up a steep concentration gradient. Under normal
conditions, a thermodynamic reaction cycle for GAT-1 in-
volves coupled translocation of two Na⫹ ions, one Cl⫺ ion and
one uncharged GABA molecule (Fig. 1B) (Kanner and Schuldiner 1987). Thus GABA is carried up its concentration gradient using the energy from the inward Na⫹ gradient and the
flow of one positive charge down the electrical gradient.
There is no doubt of the validity of the traditional view that
transporters are important for neurotransmitter clearance and
recycling after vesicular release. For example, their location is
appropriate, with GABA transporters concentrated on presynaptic terminals and peri-synaptic glial membrane (Radian et al.
1990; Ribak et al. 1996). In addition, blocking GABA or
glutamate transporters can prolong inhibitory or excitatory
postsynaptic currents (IPSCs or EPSCs) (Fig. 2, A and B)
(Dingledine and Korn 1985; Isaacson et al. 1993; Overstreet et
al. 1999; Roepstorff and Lambert 1992; Thompson and Gahwiler 1992), and blockade of GABA transporters increases
extracellular [GABA] as measured with a sniffer patch (Isaacson et al. 1993). Direct evidence for uptake after synaptic
neurotransmitter release has also been obtained using measurement of GABA and glutamate transporter currents in glia (Fig.
2, C and D) (Bergles and Jahr 1997; Kinney and Spain 2002).
Despite clear support for this model of synaptic function,
there are reasons to believe that it does not provide a complete
picture of transporter function. For example, the effect of
transporter antagonists on IPSCs and EPSCs can be small,
especially in response to low-frequency stimulation (Isaacson
et al. 1993; Nusser and Mody 2002; Overstreet and Westbrook
2003; Overstreet et al. 1999; Rossi and Hamann 1998). Glutamate transporter blockade has relatively little effect on
AMPA receptor-mediated glutamatergic EPSCs induced by a
single stimulus unless cells are first treated with cyclothiazide
to reduce receptor desensitization (Overstreet et al. 1999) (Fig.
2B). Rather than simply controlling the duration of EPSCs,
there is evidence that transporters may be more important for
prevention of overspill onto extrasynaptic receptors and
crosstalk to neighboring neurons (Overstreet and Westbrook
2003; Rossi and Hamann 1998). Transporters are also present
in locations where they would not be involved in termination of
synaptic transmission, such as on extrasynaptic membranes.
For example, in the optic nerve, a white matter tract without
synapses, GABA transporters are located on oligodendrocytes
and GABA receptors are present on axons (Sakatani et al.
Richerson, George B. and Yuanming Wu. Dynamic equilibrium of
neurotransmitter transporters: Not just for reuptake anymore. J Neurophysiol 90: 1363–1374, 2003; 10.1152/jn.00317.2003. Many electrophysiologists view neurotransmitter transporters as tiny vacuum
cleaners, operating continuously to lower extracellular neurotransmitter concentration to zero. However, this is not consistent with their
known behavior, instead only reducing extracellular neurotransmitter
concentration to a finite, nonzero value at which an equilibrium is
reached. In addition, transporters are equally able to go in either the
forward or reverse direction, and when they reverse, they release their
substrate in a calcium-independent manner. Transporter reversal has
long been recognized to occur in response to pathological stimuli, but
new data demonstrate that some transporters can also reverse in
response to physiologically relevant stimuli. This is consistent with
theoretical calculations that indicate that the reversal potentials of
GABA and glycine transporters are close to the resting potential of
neurons under normal conditions and that the extracellular concentration of GABA is sufficiently high when the GABA transporter is at
equilibrium to tonically activate high-affinity extrasynaptic GABAA
receptors. The equilibrium for the GABA transporter is not static but
instead varies continuously as the driving force for the transporter
changes. We propose that the GABA transporter plays a dynamic role
in control of brain excitability by modulating the level of tonic
inhibition in response to neuronal activity.
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G. B. RICHERSON AND Y. WU
⫹
1992). GAT-1 mRNA has also been detected in non-GABAergic neurons (Borden 1996; Frahm et al. 2000; Swan et al.
1994), and GAT-1 immunoreactivity is located on some
postsynaptic dendrites (Sagiv et al. 2002). Glutamate transporters are also present on presynaptic GABA terminals, where
they are important for supplying substrate for GABA synthesis
(Sepkuty et al. 2002). None of these observations suggest that
the traditional model is not true, but they do indicate that
neurotransmitter transporters may play additional roles in brain
function beyond termination of postsynaptic responses and
recovery of synaptically released neurotransmitter.
temperature. It should be noted that when ⌬␮˜ ⫽0 (i.e., X is at
equilibrium), this equation reduces to the Nernst equation.
For a symporter like GAT-1, all of the co-transported molecules are coupled to each other as they cross the membrane
(i.e., they are not independent). Therefore the total electrochemical driving force for GAT-1, ⌬␮˜ GAT-1, is the sum of the
linked contributions from each co-transported molecule (Aronson et al. 2003). Because one thermodynamic reaction cycle for
GAT-1 involves coupled translocation of two sodium ions, one
chloride ion and one uncharged GABA molecule, this is quantified as
⌬ ␮˜ GAT⫺1 ⫽ 2 䡠 共⌬⌿ Na ⫹ ⌬G Na兲 ⫹ 1 䡠 共⌬⌿Cl ⫹ ⌬GCl兲 ⫹ 1 䡠 共⌬GGABA兲
NONVESICULAR GABA RELEASE CAN OCCUR VIA
REVERSAL OF THE GABA TRANSPORTER
One of the properties of neurotransmitter transporters that is
not taken into account in traditional models of synaptic function is that they can reverse and release neurotransmitter (Attwell et al. 1993; Levi and Raiteri 1993). This reversal is a
direct and expected consequence of the dependence of transporters on the transmembrane ion gradients. Just as is the case
for ion channels, substrate flux through transporters is determined by the transmembrane electrochemical potential (⌬␮˜ ),
which is the sum of the electrical potential (⌬⌿) and chemical
potential (⌬G). For a single molecule X, the driving force is
quantified as
⌬ ␮˜ X ⫽ ⌬⌿ X ⫹ ⌬G X ⫽ zX 䡠 F 䡠 Em ⫹ R 䡠 T 䡠 ln
关X兴in
关X兴out
where zx ⫽ the valence of X, F ⫽ Faraday’s constant, Em ⫽
membrane potential, R ⫽ universal gas constant, and T ⫽
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and when ⌬␮˜ GAT-1 ⫽ 0, this equation reduces to
Em ⫽ ⫺
冋
冉
关GABA兴in
关Na⫹兴in
R䡠T
䡠 ln
䡠
共2 䡠 ZNa ⫹ ZCl兲F
关GABA兴out 关Na⫹兴out
冊
2
䡠
关Cl⫺兴in
关Cl⫺兴out
册
which defines the reversal potential for the transporter. It
should be noted that this latter equation has a very similar form
as the Goldman-Hodgkin-Katz equation for an ion channel.
However, there are two major differences. First, if an ion
channel is permeable to an uncharged molecule like GABA,
that molecule does not contribute to the G-H-K equation.
Second, if an ion channel is permeable to more than one ion,
each ion moves independently of the others (i.e., the movement
of each one is dependent only on the driving force for that ion).
In contrast, for a symporter like GAT-1, all co-transported
molecules will cross the membrane together, requiring that
each of their driving forces summate to give a total driving
force for all of them together. When there are two molecules of
one ion (e.g., Na⫹) that move together, each one contributes to
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1. GABA transporters are members of the Na⫹-and Cl⫺-dependent transporter family. A: evolutionary tree of mammalian
Na - and Cl⫺-dependent transporters. The GABA transporter subfamily includes transporters of GABA (GAT-1, GAT-2, GAT-3,
and GAT-4), taurine (TAUT), betaine (BETAT), and creatine (CRET). The monoamine transporter subfamily includes transporters
of serotonin (SEROT), norepinephrine (NORAT), and dopamine (DOPAT). The amino acid transporter subfamily includes
transporters of glycine (GLYT1 and GLYT2) and proline (PROT). The “orphan” transporter subfamily (identified by sequence
homology, substrate unknown) includes renal osmotic stress-induced transporter (ROSIT), neurotransmitter transporter rB21a
(NTTB21) and neurotransmitter transporter 4 (NTT4). D, dog; R, rat; M, mouse; and H, human. Adapted from Nelson (1998). B:
the stoichiometry of GAT-1 has been defined. A thermodynamic reaction cycle involves coupled translocation of 2 Na⫹ ions, one
Cl⫺ ion and 1 GABA molecule. Thus GABA is normally driven up its concentration gradient using the energy stored in the Na⫹
and electrical gradients.
FIG.
DYNAMIC EQUILIBRIUM OF NEUROTRANSMITTER TRANSPORTERS
⫹
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⫺
the concentrations of Na , Cl , and GABA were chosen to
approximate those in the normal hippocampus. These values
resulted in a calculated reversal potential of – 62 mV, which is
close to the normal resting potential of neurons. This may seem
surprising, but is actually what should be expected because the
GABA transporter would be likely to reach its equilibrium
under normal conditions. From this control curve, it can be
seen that as long as the membrane potential is more negative
than the reversal potential, the transporter will operate in the
inward direction and cells will take up GABA (forward transport). However, if the membrane potential is above the reversal
potential, then the transporter will reverse and release GABA
(reverse transport). Just like an ion channel, the driving force is
the driving force. Despite these quantitative differences in the
form of the equations for the transmembrane electrochemical
potential for ion channels and transporters, the calculated driving force for a transporter can be viewed in the same way as the
driving force for an ion channel. In other words, there will be
no net substrate flux when membrane potential is equal to the
reversal potential, and when it is not there will be a driving
force generated to move solute in the direction needed to return
membrane potential back to the reversal potential.
The solution to the equation for ⌬␮˜ GAT-1 is shown in Fig.
3A, under three different conditions. Under control conditions,
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FIG. 3. The direction and rate of GABA transport is determined by the
combined chemical and electrical potential difference for all of the co-transported substrates. For GAT-1, this total transmembrane electrochemical potential, ⌬␮˜ GAT-1, or driving force, is dependent on the membrane potential and
the transmembrane gradients for sodium, chloride, and GABA. Based on the
equations in the text, this driving force is plotted for 3 conditions, as illustrated
at the top. Control conditions were chosen to approximate the normal concentrations of the substrates inside and outside of a hippocampal neuron. The
calculated reversal potential under these conditions (⫺62 mV) was close to the
normal resting potential of neurons. As would be the case for an ion channel,
there would be net influx of substrate (forward transport) when the membrane
potential was more negative than the reversal potential, and there would be net
efflux (reverse transport) when the membrane potential was more positive. An
increase in intracellular [Na⫹] to 30 mM led to a negative shift in the reversal
potential to –99 mV, so that there would be reverse transport at normal resting
potential. An increase in extracellular [GABA] to 10 ␮M led to a positive shift
in the reversal potential to ⫹60 mV, so that there would be a large driving
force for GABA uptake at all physiologically relevant membrane potentials.
Vertical dotted line highlights the reversal potential for the control conditions,
and the direction of the arrows in the 3 schematics at top represent the direction
of substrate flux when the membrane potential is equal to – 62 mV.
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FIG. 2. GABA and glutamate transporters terminate postsynaptic responses
by removing neurotransmitter from the synaptic cleft. A: presynaptic stimulation of a hippocampal GABAergic neuron induces an inhibitory postsynaptic
current (IPSC) in a pyramidal cell (top). The GABA transporter antagonist
tiagabine causes prolongation of the IPSC (bottom). Adapted from Thompson
and Gahwiler (1992). B: the glutamate transporter antagonist L-trans-pyrrolidine-2,4-dicarboxylate (L-trans-PDC) has no effect on an excitatory postsynaptic current (EPSC) induced in a cerebellar granule cell (top; control and
L-trans-PDC responses are superimposed). Treatment with cyclothiazide decreases desensitization and prolongs the EPSC (bottom). After cyclothiazide,
L-trans-PDC further prolongs the EPSC [from Overstreet et al. (1999)]. C:
presynaptic stimulation in the neocortex induces a current in astrocytes that is
partially blocked by the GAT-1 inhibitors NO-711 and SKF-89976a (top). The
difference between these 2 curves (bottom) is the transporter current induced
by uptake into glia of synaptically released GABA [from Kinney and Spain
(2002)]. D: stimulation of Schaffer collateral fibers induces a current in glia in
hippocampal slices. This is a transporter current induced by reuptake of
synaptically released glutamate, because it is blocked by the glutamate transporter antagonist D,L-threo-3-hydroxyaspartate [THA; from Bergles and Jahr
(1997)].
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FIG. 4. The GABA transporter can reverse. A: GABA release was measured from cultured striatal neurons using HPLC. GABA release was increased
above basal levels by an increase in [K⫹]o to 56 mM (K⫹). This was only
partially inhibited by tetanus toxin pretreatment, by 0 calcium bath solution or
by a combination of both. When GABA release was induced by veratridine
(Ver; 20 ␮M) or glutamate (Glu; 100 ␮M), it was not affected by tetanus toxin
and/or 0 calcium solution. These latter 2 stimuli are thought to lead to a larger
increase in intracellular [Na⫹] than when depolarization is induced by K⫹.
Thus these results are consistent with enhancement of GABA transporter
reversal by an increase in intracellular [Na⫹] [adapted from Pin and Bockaert
(1989)]. B: transporter currents measured in Xenopus oocytes expressing
GAT-1. The direction of current is the same as the direction of Na⫹ and GABA
flux. Thus at membrane potentials below the reversal potential (⫹45 mV),
there is net influx of GABA, whereas at potentials above the reversal potential
there is net efflux of GABA (reverse transport). These experiments also
demonstrated that the measured reversal potential was approximately equal to
theoretical calculations of reversal potential [from Lu and Hilgemann (1999)].
C: heteroexchange GABA release. When 1 molecule of nipecotic acid is
applied to the extracellular face of a membrane, it induces the release of one
molecule of GABA. The molecular mechanism is unclear, but is usually
schematically depicted as shown.
2002). However, the majority of electrophysiological and biochemical assays have indicated that GABA transporters operate with a fixed stoichiometry under most physiological conditions, and their behavior, including passive reversal, is accurately predicted by the equations shown above. For example,
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determined by the difference between the membrane potential
and the reversal potential. It should be noted that the driving
force is expressed in kilocalories per mole of substrate transported across the membrane. This driving force can be converted using appropriate constants to an equivalent electrical
potential (in millivolts) as is the convention for the driving
force for an ion channel.
As expected, the reversal potential varies depending on the
local prevailing conditions, and is sensitive to the sodium and
GABA gradients. When intracellular [Na⫹] was doubled to 30
mM, this caused a shift in the reversal potential to –99 mV
(Fig. 3). This example illustrates two important points: an
increase in intracellular [Na⫹] can lead to net efflux of GABA
at resting potential and the GABA transporter is more sensitive
than an ion channel to changes in the sodium gradient (the
corresponding change in Nernst potential for Na⫹ would only
be 18 mV). This is due to the coupled translocation of two
sodium ions.
When extracellular [GABA] was increased 100-fold, there
was a shift in reversal potential to ⫹60 mV (Fig. 3). Thus
unlike an ion channel, a change in the gradient of an uncharged
molecule leads to a shift in reversal potential, and this shift was
⬃60 mV with each 10-fold change in the gradient. This dependence of reversal potential on the GABA gradient is due to
the fact that there is obligatory co-transport of one net positive
charge with each GABA molecule. This example illustrates
that a sufficiently large increase in extracellular [GABA] can
lead to net influx of GABA at all physiologically relevant
membrane potentials, so that as expected there would be net
uptake of extracellular GABA in response to synaptic GABA
release. However, the transporter could still reverse given
sufficiently strong depolarization.
There have been many experimental studies that have confirmed that the GABA transporter will reverse, in most cases
using biochemical measures of GABA release. For example,
after removal of extracellular Ca2⫹, GABA release still occurs
from cortical slices in response to an increase in K⫹ to 50 mM,
to exposure to 50 ␮M veratridine, or to high-frequency electrical stimulation (Szerb 1979). Similarly, GABA release still
occurs in the absence of extracellular Ca2⫹ from cultured
striatal neurons (Pin and Bockaert 1989) and from cultured
cortical neurons (Belhage et al. 1993) in response to 55–56
mM K⫹ or 100 ␮M glutamate. In fact, GABA release can even
occur after treatment with tetanus toxin combined with removal of extracellular Ca2⫹ (Fig. 4A). This GABA release is
due to transporter reversal because it is blocked by GABA
transporter antagonists (Belhage et al. 1993; Pin and Bockaert
1989).
GABA transporter reversal has also been measured directly
using electrophysiological recordings from GABA transporters. For example, GAT-1 transporter currents reverse in response to depolarization (Fig. 4B) or an increase in intracellular Na⫹ (Lu and Hilgemann 1999). Similar experiments have
revealed that the GABA transporter can sometimes operate in
an “uncoupled” manner, whereby the stoichiometry is not
fixed, instead diverging from the normal ratio of 2 Na⫹:1 Cl⫺:1
GABA (Cammack et al. 1994). This slippage is generally
believed to only occur under artificial conditions and be an
imperfection in the design of transporters; but it has been
suggested that slippage may actually play an important role as
a safety valve in dissipating excess driving force (Nelson et al.
DYNAMIC EQUILIBRIUM OF NEUROTRANSMITTER TRANSPORTERS
TABLE 1. Differences between vesicular and transporter-mediated
GABA release
Vesicular Release
Transporter Reversal
Cell depolarization
Low calcium
Tetanus or
botulinum toxin
Rise in [Na⫹]i
Energy depletion
Source of GABA
Location of pool
Nipecotic acid
Enhances
Blocks
Blocks
Enhances
No effect
No effect
No effect
Reduces
Neurons only
Synaptic vesicles
Prolongs IPSPs
Noncompetitive
transport blockers
Prolong IPSPs
Enhances
Enhances
Neurons and glia
Cytosol
Induces release, then
blocks
Block
the validity of these calculations has been experimentally verified for both the glutamate (Zerangue and Kavanaugh 1996)
and GABA transporters (Lu and Hilgemann 1999).
GABA transporter reversal can also be induced pharmacologically by nipecotic acid. This compound is a GABA analogue that induces a phenomenon referred to as heteroexchange
release (Honmou et al. 1995; Solis and Nicoll 1992; Szerb
1982a). Nipecotic acid does not directly activate GABA receptors. However, it indirectly induces GABA receptor-mediated
currents when applied to cultures or brain slices by causing
nonvesicular release of GABA from neighboring cells. This
release is blocked by antagonists of GABA transporters. The
molecular mechanism of heteroexchange GABA release has
not been well defined but is widely believed to be due to the
exchange of one intracellular GABA molecule for one extracellular nipecotic acid molecule (Fig. 4C). Continued application of nipecotic acid later causes GABA transporter blockade.
These dual effects of this drug can sometimes make it difficult
to interpret a response to its application.
In summary, there is a large body of experimental data
showing that GABA transporters can reverse, and there is a
solid theoretical framework for explaining why this can happen. Table 1 compares the conditions that induce vesicular
release with those that induce transporter-mediated release.
THE GABA TRANSPORTER REVERSES
SURPRISINGLY EASILY
The stimuli used to induce GABA transporter reversal in
many previous studies would not occur under physiological
conditions (e.g., 55 mM K⫹ or 100 ␮M glutamate). The use of
such strong stimuli has led many to assume that the GABA
transporter won’t reverse without a pathologically large stimulus. However, this is not the case, and the use of strong stimuli
was probably a result of the relative insensitivity of the biochemical assays used to measure GABA release. As suggested
by the calculations in Fig. 3, the reversal potential for GAT-1
would be expected to be close to the normal resting potential of
neurons, so that a small level of depolarization would cause
reversal. This is supported by some early experiments that
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clearly demonstrated that GABA transporters can reverse under physiologically relevant conditions. For example, calciumindependent GABA release can be induced by electrical stimulation of slices from the rat cortex (Szerb 1979) and striatum
(Bernath and Zigmond 1988). In the fish retina, depolarization
of GABAergic horizontal cells, which are deficient in synaptic
vesicles, induces a postsynaptic response in bipolar cells
(Schwartz 1987). This response is due to GABA release via
reversal of the GABA transporter because it is not dependent
on calcium and is blocked by nipecotic acid.
We directly tested the hypothesis that a small depolarizing
stimulus could induce reversal of GABA transporters from the
mammalian hippocampus and that the resulting nonvesicular
GABA release leads to neuronal inhibition (Gaspary et al.
1998; Wu et al. 2001). Depolarization was induced by an
increase in [K⫹]o from 3 to 6 –12 mM. This stimulus is physiologically relevant, since [K⫹]o increases during neuronal
firing in vivo. For example, an increase in [K⫹]o to ⬎12 mM
occurs in the rat hippocampus in vivo in response to electrical
stimulation of the angular bundle (Somjen and Giacchino
1985). The method chosen to assay GABA release was to use
electrophysiological recordings from neurons because this is a
very sensitive way of detecting the component of GABA
release that is functionally relevant (Fig. 5A). Patch-clamp
recordings were made from rat hippocampal neurons in culture
under conditions designed to block all ion channels in the
recorded neuron except GABAA receptors (Gaspary et al.
1998; Wu et al. 2001). Recordings were made in the absence of
extracellular Ca2⫹ or after pretreating neurons with tetanus
toxin, both of which are widely used to block vesicular GABA
release. An increase in [K⫹]o to 12 mM led to a large chloride
current. This current was due to activation of GABAA receptors because it was blocked by bicuculline (Fig. 5B) and
picrotoxin. The GABA release that caused the activation of
GABA receptors was due to transporter reversal because it was
blocked by SKF-89976a and NO-711, two GAT-1 antagonists.
An increase in [K⫹]o to as little as 6 mM was sufficient to
induce transporter reversal (Wu et al. 2001). These results
demonstrate that even a small increase in [K⫹]o induces GABA
transporter reversal, increasing ambient [GABA] and inducing
neuronal inhibition.
The experiments described here are consistent with the calculations of Fig. 3 predicting that the reversal potential of
GAT-1 is close to its equilibrium under normal resting conditions. In addition, when neurons increase their firing rate, there
is an increase in cytosolic [Na⫹], which would cause a negative
shift in the reversal potential (Fig. 3). When combined with the
increase in membrane potential (due to firing as well as increased extracellular [K⫹]) (Somjen and Giacchino 1985), this
would lead to a large outward driving force for GABA efflux.
For these reasons, reversal of GABA transporters would be
greatest during high-frequency firing. These predictions should
be placed in context with the normal role of the GABA
transporter in reuptake of synaptic GABA. During an IPSP,
[GABA] within the synaptic cleft rises to near 1 mM (Jones
and Westbrook 1995; Mozrzymas et al. 1999). This would lead
to a theoretical reversal potential of ⫹182 mV, assuming all
other values are the same as for the control example in Fig. 3.
Thus the increase in synaptic [GABA] during an IPSP would
lead to such a large driving force that reuptake would still
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Energy depletion leads to a decrease in the ability to repackage vesicles but
enhances transporter-mediated GABA release due to breakdown of the Na⫹
gradient and membrane potential. Nipecotic acid induces transporter reversal
via heteroexchange followed by blockade of release and reuptake. Noncompetitive transport blockers (e.g. NO-711 and SKF-89976a) only block release
and reuptake.
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occur at all physiologically relevant membrane potentials, consistent with a role of transporters in terminating IPSPs.
GABA TRANSPORTER REVERSAL IS ENHANCED
BY TWO NEW ANTICONVULSANTS
Many anticonvulsant drugs prevent seizures by increasing
GABAergic inhibition, such as by potentiating GABA receptors (e.g., benzodiazepines, barbiturates) or blocking reuptake
(e.g., tiagabine). Recently, we have shown that two new anticonvulsant drugs, gabapentin and vigabatrin, enhance GABA
transporter reversal. Gabapentin was designed as a lipophilic
GABA agonist but has no direct effect on GABA receptors or
the GABA transporter. However, it does increase brain GABA
levels in humans in vivo as measured using magnetic resonance spectroscopy (Petroff et al. 1996). Using patch-clamp
recordings from rat hippocampal slices (Honmou et al. 1995)
as well as sucrose gap recordings from rat optic nerve (Kocsis
and Honmou 1994), it was found that gabapentin increases
heteroexchange GABA release induced by nipecotic acid (Fig.
4C). This observation provided the first electrophysiological
evidence for an effect of gabapentin on GABAergic inhibition.
Interestingly, gabapentin also leads to an increase in expression
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⫹
FIG. 5. An increase in [K ]o causes depolarization, which induces an
increase in ambient [GABA]. A: patch-clamp recordings of GABAA receptormediated currents were used to assay GABA release. Experiments were
performed in 0 calcium bath solution. When the GABA transporter reversed,
GABA release was detected as a change in holding current in the recorded
neuron. B: application of 12 mM K⫹ (bar) caused GABA release that induced
an inward current (left). This current was due to GABAA receptor activation
because it was blocked by bicuculline (middle). The GABA release was due to
GAT-1 reversal because it was blocked by SKF-89976a and NO-711 (not
shown) [from Gaspary et al. (1998)].
of GABA transporters on the cell surface (Whitworth and
Quick 2001). It is unclear whether these two observations are
related, or if one causes the other. However, it is conceivable
that either an increase in number of functional GABA transporters increases the flux of GABA when transporter reversal
occurs or an increase in ambient [GABA] due to enhanced
transporter reversal leads to an increase in surface expression
of transporters (Bernstein and Quick 1999). These findings
clearly link the GABA transporter with gabapentin, a widely
used medication whose mechanism has eluded definition.
Vigabatrin is an anticonvulsant whose biochemical mechanism has been well defined, but whose electrophysiological
mechanism has remained unclear. This drug works as a “suicide substrate” for GABA transaminase, binding irreversibly to
inhibit the enzyme. Recovery requires synthesis of new protein. As would be predicted, blocking GABA transaminase
induces an increase in brain GABA levels (Rothman et al.
1993; Schechter et al. 1977) and also enhances GABA release
(Gram et al. 1988; Szerb 1982b). It has been widely assumed
that the increase in GABA would enhance inhibitory synaptic
transmission. However, there has been no direct evidence for
an increase in inhibition, and in fact vigabatrin can cause a
decrease in synaptic inhibition (Jackson et al. 1994; Overstreet
and Westbrook 2001). To define the electrophysiological
mechanism of this drug, we made patch-clamp recordings from
neurons in rat hippocampal cultures pretreated with 100 ␮M
vigabatrin. There was no effect of vigabatrin on the amplitude
of spontaneous miniature IPSCs, and there was a small paradoxical decrease in the amplitude of evoked IPSCs during
paired recordings (Wu et al. 2003). In contrast to the lack of an
increase in this “phasic” inhibition, there was a marked enhancement of “tonic” GABAergic inhibition due to an increase
in ambient [GABA] (Wu et al. 2001, 2003). The tonic inhibition was not prevented by calcium-free bath solution or tetanus
toxin pretreatment (Fig. 6, A and B) (Wu et al. 2001, 2003).
During perforated-patch recordings, the increase in ambient
[GABA] was prevented with NO-711 and SKF-89976a, two
GAT-1 specific antagonists (Wu et al. 2001, 2003). Thus the
increase in ambient [GABA] was due to continuous GABA
efflux via transporter reversal. These results, together with the
equations shown above, suggest that the increase in cytosolic
[GABA] induced by both gabapentin and vigabatrin causes the
reversal potential for the GABA transporter to become more
negative. This could in turn lead to GABA transporter reversal
even at normal resting potential.
One of the most interesting results from these experiments
was that when patch-clamp recordings were made in whole cell
mode, GAT-1 blockade caused an increase in ambient [GABA]
(Fig. 6C), which is opposite the response seen when using
perforated patch recordings (Wu et al. 2003). Thus whole cell
recordings can lead to a conclusion about the role of the GABA
transporter that is exactly opposite that reached when perforated-patch recordings are used. The reason for this artifact
induced by whole cell recording is unclear, but it could be
prevented simply by adding Na⫹ and GABA to the whole cell
electrode solution (Fig. 6D). The simplest explanation is that
there is a loss of Na⫹ and GABA from the recorded neuron
during whole cell recordings. However, it is possible that some
other factor is lost from cells, but Na⫹ and GABA in the
pipette solution lead to normalization of the response. There
are some reasons to believe that this latter possibility might be
DYNAMIC EQUILIBRIUM OF NEUROTRANSMITTER TRANSPORTERS
the case (Wu et al. 2003). Despite the inability to define the
mechanism, it is critically important that this artifact has been
recognized to exist. There have been many recent studies that
have used the response to GAT-1 blockade as the sole evidence
to rule out GABA transporter reversal as the mechanism of
tonic inhibition. However, these studies have used whole cell
recordings without Na⫹ or GABA in the electrode solution. It
is now clear that caution should be used in interpreting results
from GAT-1 blockade when using whole cell recordings.
The potency of the effect of vigabatrin on hippocampal
cultures suggests that the increase in ambient [GABA] observed in vitro is likely to be related to the anticonvulsant effect
of the drug in vivo. As little as 50 nM vigabatrin was sufficient
to cause a significant increase in ambient [GABA] in hippocampal cultures (Fig. 7A). It has been difficult to determine
what concentration of the drug should be used in vitro to mimic
clinically relevant conditions in vivo. Peak serum levels of
vigabatrin exceed 100 ␮M after chronic dosing with 1–2 g/day
in humans (Ben-Menachem 1995), but there is little information on brain tissue concentrations. The response to the drug is
not dependent on peak levels like most reversible receptor
antagonists but is instead proportional to the product of average
concentration and duration of exposure (Jung and Palfreyman
1995). Thus peak serum levels may be less relevant to the
anticonvulsant effect than the average tissue concentration
during chronic use. The ability of 50 nM vigabatrin to increase
J Neurophysiol • VOL
ambient [GABA] in culture indicates that much lower concentrations of the drug are effective than those suggested by the
peak serum levels.
The time course of the effect of vigabatrin in vitro also
suggests that the increase in ambient [GABA] is relevant to the
anticonvulsant effect in vivo. Vigabatrin can directly inhibit
the GABA transporter, but this effect is immediate and is
reversible on washout (Jung and Palfreyman 1995). In contrast,
tonic inhibition induced by vigabatrin in vitro required 4 –5
days to reach a maximum and was sustained for hours after
washout of the drug (Fig. 7B). This slow onset and lingering
effect is consistent with slow inactivation of GABA transaminase followed by a further delay in the increase in cytosolic
[GABA]. This temporal profile reflects the anticonvulsant effect of the drug in vivo. After a single intraperitoneal injection
of vigabatrin, the elevation in brain GABA levels and the
depression of GABA transaminase activity both last for ⬎4
days (Jung and Palfreyman 1995). Total GABA levels peak
within 12 h, but the GABA pool associated with nerve terminals doesn’t peak until 60 h, and it is the latter that correlates
with the peak anticonvulsant effect (Gale and Iadarola 1980).
Thus both the potency of the drug and the temporal profile
FIG. 7. Concentration and time dependence of the effect of vigabatrin on
tonic inhibition. Summary of data from experiments like that in Fig. 6A. A:
tonic inhibition was induced after 3– 4 days of treatment with as little as 50 nM
vigabatrin, and was concentration dependent. B: the tonic inhibition induced by
vigabatrin (100 ␮M) was very slow to develop, requiring ⬎4 days to reach a
maximum. For both parts, all data points for vigabatrin-treated neurons are
significantly greater than control data points [P ⬍ 0.001; from Wu et al.
(2001)].
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FIG. 6. Vigabatrin causes tonic inhibition of cultured hippocampal neurons
due to spontaneous GABA transporter reversal. Same methods as shown in
Fig. 5A. A: after treatment with 100 ␮M vigabatrin for 4 days, a large tonic
GABAA receptor-mediated current was induced in neurons. The size of the
tonic current was approximately the same in 0 calcium Ringer (black trace)
compared with normal Ringer (gray trace). B: the tonic current was also not
blocked by pretreatment with tetanus toxin. C: when whole cell recordings
were made without GABA or Na⫹ in the electrode solution, the response to
SKF-89976a was opposite that of the response to bicuculline. This was
interpreted as indicating that the GABA transporter was working in the
forward direction and that the release of GABA that caused the increase in
tonic current was due to some mechanism other than GABA transporter
reversal. D: when the same experiment was performed with 20 mM GABA and
20 mM Na⫹ in the electrode solution, the tonic current was blocked by
SKF-89976a. In this case, the opposite conclusion was reached: that the
increase in ambient [GABA] was due to GABA transporter reversal. The result
shown in D was also obtained when using perforated-patch recordings. Thus
the whole cell recording technique can lead to an artifactual response to GAT-1
blockade unless GABA and Na⫹ are added to the electrode solution. Bicuc,
bicuculline; SKF, SKF-89976a. A is from Wu et al. (2001). B–D are from Wu
et al. (2003).
1369
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G. B. RICHERSON AND Y. WU
suggest that the anticonvulsant effect of the drug is due to the
increase in tonic inhibition.
AMBIENT GABA AND TONIC INHIBITION
J Neurophysiol • VOL
ROLE OF THE GABA TRANSPORTER IN TONIC
INHIBITION
There has been relatively little focus on the role of the
GABA transporter in regulating the level of tonic inhibition.
The standard approach to determine whether GABA transporter reversal contributes to tonic inhibition is to apply GABA
transporter antagonists and see whether tonic inhibition increases or decreases. However, as discussed in the preceding
text, the existence of an artifact when using whole cell recordings was not previously recognized, so that some of the previous work using this approach may have reached an erroneous
conclusion about the role of GABA transporter reversal in
tonic inhibition. In addition, it is not necessary for GABA
transporters to reverse to play an important role in regulating
tonic inhibition. GABA transporters do not function like vacuum cleaners, working continually in the forward direction to
reduce [GABA]o to zero. Instead, they try to maintain ambient
[GABA] at their set point (i.e., the reversal potential). If the set
point was sufficiently low, it would be likely that tonic inhibition would be low even if there was a large amount of
vesicular GABA release. If the set point was high, then ambient [GABA] and tonic inhibition would be high even if there
was only a small amount of vesicular GABA release. Under
both conditions, the transporter may still operate in the forward
direction to take up GABA released by vesicular fusion. However, an increase in the set point would result in a higher
ambient [GABA] even if the amount of vesicular GABA
release is small. Therefore, instead of focusing on the direction
of GABA flux, an alternative way to view the role of the
GABA transporter is to calculate the [GABA]o when the transporter is at equilibrium, and assume that the transporter is
maintaining [GABA]o close to this equilibrium. This is a valid
assumption, supported by the calculations in Fig. 3 for control
conditions, which show that the reversal potential is near the
normal resting potential.
We rearranged the equation for the reversal potential of
GAT-1 (see preceding text) to solve for extracellular [GABA]
instead of membrane potential (Wu et al. 2003). These calculations predicted that ambient [GABA] would be high enough
(ⱖ0.1 ␮M) under physiological conditions to activate highaffinity GABAA receptors. At a membrane potential of ⫺60
mV, and a [Na⫹]i of 13 mM, [GABA]o at equilibrium would be
0.1 ␮M if cytosolic [GABA] is 2.5 mM (Fig. 8A). Although it
is difficult to precisely measure free cytosolic [GABA], this
value is consistent with experimentally measured [GABA] in
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Until recently, GABAergic inhibition was believed to be
mediated solely by phasic IPSPs due to vesicular GABA release. During the last decade, it has been recognized that there
is also a tonic form of inhibition like that seen after vigabatrin
treatment, but occurring spontaneously (i.e., without drug treatment). This tonic inhibition is due to an ambient [GABA] that
is sufficiently high to continuously activate GABAA receptors
on some neurons (Bai et al. 2001; Brickley et al. 1996, 2001;
Liu et al. 2000; Nusser and Mody 2002; Otis et al. 1991; Rossi
and Hamann 1998; Stell and Mody 2002; Vautrin et al. 2000;
Wall and Usowicz 1997; Yeung et al. 2003). The magnitude of
this tonic inhibition can be surprisingly high with the total
charge transfer greatly exceeding that due to phasic GABA
release (Brickley et al. 1996; Nusser and Mody 2002; Wall and
Usowicz 1997). The specific role of tonic inhibition is unknown, but it would clearly have a large effect on brain
excitability. The shunt current may also contribute to “gain
modulation” or responsiveness to synaptic inputs (Chance et al.
2002).
Tonic GABAergic inhibition results from activation of extrasynaptic, high-affinity GABAA receptors that do not desensitize (reviewed in Mody 2001). The evidence that the two
types of inhibition are mediated by different types of GABA
receptor comes in part from the observation that tonic inhibition and phasic IPSCs can be pharmacologically separated.
NO-711 and midazolam selectively enhance tonic inhibition
(Nusser and Mody 2002; Yeung et al. 2003), whereas zolpidem
selectively enhances phasic inhibition (Nusser and Mody
2002). A low concentration of gabazine blocks phasic inhibition without affecting tonic inhibition, whereas higher concentrations also block tonic inhibition (Stell and Mody 2002;
Yeung et al. 2003).
Ambient [GABA] has been measured to be 0.2– 0.8 ␮M in
the rat hippocampus (Lerma et al. 1986; Tossman et al. 1986).
This [GABA] is sufficient to activate extrasynaptic GABAA
receptors but not GABAA receptors within the synaptic cleft.
The specific subunit composition of high-affinity, extrasynaptic GABAA receptors is unclear, but ␦ subunits are only found
in extrasynaptic membrane (Nusser et al. 1998). There is
preferential assembly of the ␦ subunit with the ␣6 subunit, and
GABAA receptors with the subunit composition of ␣6␤2␦ or
␣6␤3␦ have a high affinity for GABA (EC50 ⫽ 0.19 – 0.27 ␮M)
(Saxena and Macdonald 1996) and also have greatly reduced
desensitization (Saxena and Macdonald 1994). These are precisely the properties expected of GABA receptors involved in
tonic inhibition, and in fact GABAA receptors containing the
␣6 subunit appear to contribute to tonic inhibition in cerebellar
neurons (Rossi and Hamann 1998). This is consistent with the
finding that tonic GABAergic inhibition is absent in the cerebellum of mice lacking expression of extrasynaptic ␣6␤X␦
receptors due to targeted deletion of the ␣6 gene (Brickley et al.
2001). Although the ␣6 and ␦ subunits appear to be responsible
for tonic inhibition in the cerebellum, the ␣6 subunit is limited
within the brain primarily to cerebellar granule cells (Jones et
al. 1997). There is less known about the subunit composition of
GABAA receptors that contribute to tonic inhibition in the
hippocampus and elsewhere (Mody 2001).
A variety of other mechanisms may also be involved in
producing tonic inhibition, including a continuous barrage of
spontaneous miniature IPSCs that summate with each other
(Otis et al. 1991), “spillover” of GABA out of synapses onto
extrasynaptic membrane (Brickley et al. 1996; Rossi and Hamann 1998), detachment of GABA from binding sites on a
surface matrix that becomes exposed during exocytosis (Vautrin et al. 2000), release of GABA from astrocytes (Liu et al.
2000), and constitutive activation of GABAA receptors in the
absence of GABA (Neelands et al. 1999). The relative importance of each of these mechanisms in tonic inhibition is unclear, and may vary depending on the experimental conditions.
DYNAMIC EQUILIBRIUM OF NEUROTRANSMITTER TRANSPORTERS
1371
FIG. 8. Solutions to the equation for the reversal potential of the GABA
transporter. A: effect of membrane potential on the equilibrium [GABA]o at
different levels of [GABA]i. [Na⫹]i was 13 mM. Note that when [GABA]i is
5–10 mM and Em is ⫺60 mV, the transporter is at equilibrium when [GABA]o
is high enough to activate high-affinity, extrasynaptic GABAA receptors (see
text). B: effect of membrane potential on the equilibrium [GABA]o at different
levels of [Na⫹]i. [GABA]i was 5 mM. Note that depolarization combined with
an increase in [Na⫹]i, such as would occur during a burst of firing, would lead
to a large increase in the equilibrium [GABA]o [from Wu et al. (2003)].
some cells (Otsuka et al. 1971). In fact, cytosolic [GABA] has
been reported to be 6.6 mM in Purkinje cells in the cerebellum
(Otsuka et al. 1971), a brain region in which tonic inhibition is
particularly prominent (Brickley et al. 1996; Rossi et al. 2003).
As shown in Fig. 3, the reversal potential for GAT-1 is sensitive to the GABA gradient, so the ambient [GABA] would be
expected to be higher outside neurons with a relatively high
cytosolic [GABA]. For example, cells with a cytosolic
[GABA] of 6.6 mM and a membrane potential of ⫺60 mV
would have a [GABA]o of 0.26 ␮M at equilibrium. These
calculations suggest that the GABA transporter may regulate
ambient GABA at a level high enough to cause spontaneous
tonic inhibition in some parts of the brain. When neurons
increase their firing rate, the resulting increase in membrane
potential, combined with an increase in [Na⫹]i, would lead to
an even greater increase in the equilibrium [GABA]o (Fig. 8B).
This would be particularly important during stroke or seizures
when there is a collapse of electrochemical gradients across
membranes. However, as illustrated in Fig. 8, such extreme
conditions would not be necessary for the equilibrium
[GABA]o to reach a high level. The theoretical importance of
the equilibrium value is that the GABA transporter would be
incapable of lowering ambient [GABA] below it and would in
fact reverse if [GABA]o dropped below this value.
In hippocampal cultures treated with vigabatrin, the increase
in tonic inhibition is due to GABA transporter reversal (Wu et
J Neurophysiol • VOL
SEEKING A CONSTANTLY CHANGING
EQUILIBRIUM
The solutions to the reversal potential equation shown in
Fig. 8 are based on steady-state values of membrane potential
and substrate concentrations. However, neurons in vivo are
rarely at steady state, so the equilibrium [GABA]o is constantly
changing. Therefore even though the net direction of GABA
flux through the transporter is likely to be inward, the direction
and magnitude of GABA flux would constantly vary. We
envision the following scenario. During resting conditions,
there would be only a small amount of inward GABA flux to
recover basal vesicular release. When a burst of firing occurs,
there would first be increased GABA uptake due to increased
[GABA]o. As [Na⫹]i rises, GABA uptake would slow and then
begin to reverse. This reversal would occur in a dynamic
fashion with pulses of GABA release with each depolarization.
Finally, at the end of the burst of activity there would be a large
surge of GABA reuptake to recover the GABA that was
released during the burst. Not only would transporter activity
respond dynamically to the changing conditions, but it would
also be spatially inhomogeneous, with differences in flux of
GABA among the synapse, the perisynaptic regions, and distant extrasynaptic locations. It is also now recognized that
GABAergic neurons cannot be viewed as homogeneous, with
different classes of GABAergic neurons present even within a
single brain region (Poncer et al. 2000). It is likely that the
function of GABA transporters varies among these different
types of GABAergic neurons as indicated by the recent finding
that the density of GAT-1 varies among different types of
synapses (Chiu et al. 2002).
In addition to the complexity of the system described in the
preceding text, the properties of GABA transporters themselves are also dynamic. For example, the GABA transporter
can be shuttled back and forth into the plasma membrane from
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al. 2001, 2003). However, this does not necessarily mean that
vigabatrin always causes transporter reversal. An alternative
way of viewing this finding is that vigabatrin simply increases
the equilibrium [GABA]o for the transporter (by increasing
[GABA]i). After treatment with vigabatrin in vivo, there are
two ways that tonic inhibition could increase without reversal
of the GABA transporter: by increasing the equilibrium
[GABA]o; or by decreasing the ability of the GABA transporter to reach this equilibrium by reducing the number or
efficiency of functional GABA transporters in the cell membrane (Bernstein and Quick 1999; Deken et al. 2003). As long
as there is vesicular GABA release, then either mechanism
could lead to an increase in tonic inhibition. Thus tonic inhibition is not dependent on reversal of GABA transporters, and
in fact GABA transporters would normally operate in the
forward direction to prevent excessive tonic inhibition, by
virtue of reuptake of vesicular release—precisely as predicted
by the classical view of the GABA transporter. This dichotomy
may seem counterintuitive, but these dual roles of the GABA
transporter in reducing tonic inhibition by reuptake and regulating the level of tonic inhibition via changes in the equilibrium [GABA]o, follow naturally from the biophysics of transporters. To accomplish the latter, the transporter will reverse
whenever necessary, but reversal is not a prerequisite for an
increase in [GABA]o.
1372
G. B. RICHERSON AND Y. WU
intracellular sites of sequestration in response to an increase in
extracellular [GABA] (Bernstein and Quick 1999). Interestingly, GABA transporters are also rapidly inserted into the
plasma membrane of synaptic terminals in response to presynaptic depolarization (Deken et al. 2003). These transporters are
sequestered in presynaptic vesicles that are a different subset
than those that contain GABA and are rapidly inserted into the
plasma membrane in response to a burst of activity, possibly to
maximize the efficiency of reuptake after release. Thus not
only are transporters constantly seeking an equilibrium that is
a moving target, but the transporters themselves are constantly
moving.
reverse in response to stimulation of excitatory input from the
subthalamic nucleus (Falkenburger et al. 2001). The resulting
nonvesicular dopamine release induces autoinhibition by activating dopamine receptors on nigral neurons. The serotonin
transporter can also reverse easily. For example, methylenedioxymethamphetamine (MDMA; Ecstasy) and similar
drugs induce heteroexchange serotonin release by reversal of
the serotonin transporter (Rudnick and Wall 1992). Thus it is
likely that reversal to physiological stimuli is a property common to many neurotransmitter transporters.
ALL NEUROTRANSMITTER TRANSPORTERS CAN
REVERSE
The emerging view of neurotransmitter transporters is more
complex than the traditional view. The classical model, although accurate in a global and static sense, is too simplistic to
describe subcellular and dynamic events. On average, transporters do function to terminate synaptic transmission and
scavenge transmitter for reuse. However, transporters can also
reverse, and there is strong evidence that some of them do so
in response to physiological changes in substrate gradients and
membrane potential. Thus transporters can no longer be
viewed as tiny vacuum cleaners whose sole purpose is to
continuously suck neurotransmitter completely out of synaptic
clefts.
Understanding the role of transporters in synaptic function
and in control of tonic inhibition is important because these
molecules contribute to a variety of pathological conditions
and are targets of many drugs. For example, reversal of the
GABA transporter is likely to occur during seizures, and regulation of tonic inhibition by GABA transporters may modulate seizure threshold. There are many questions that still need
to be answered about the complexities of transporter function.
Does tiagabine have an anticonvulsant effect by blocking
GABA reuptake (Thompson and Gahwiler 1992) as well as a
proconvulsant effect by blocking transporter reversal? What is
the relative role of GABA transporters on neurons versus glia?
Is GABA transporter reversal always a good thing, given that
the conditions for transporter reversal also favor the generation
of depolarizing GABA responses (Staley et al. 1995)? Are
there unrecognized mechanisms for modulation of GABA
transporters by second-messenger systems or pharmacological
agents? How many other neurotransmitter transporters share
these properties described for GABA transporters? A more
thorough understanding of the dynamic equilibrium of transporters will help to define their role in normal brain function
and pathological states.
J Neurophysiol • VOL
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