From the Biological Sciences Department, California
State Polytechnic University, Pomona, California 91768-4032, § Department of Biochemistry, The George S. Wise Faculty of
Life Sciences, Tel Aviv University, Tel Aviv 69978, Israel, and
¶ Department of Physiology, School of Medicine, University of
California, Los Angeles, California 90095
Received for publication, July 29, 2002, and in revised form, February 20, 2003
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ABSTRACT |
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Valproate is an important anticonvulsant
currently in clinical use for the treatment of seizures. We used
electrophysiological and tracer uptake methods to examine the effect of
valproate on a Valproate (2-propylpentanoate) has been in clinical use since 1967 and
is effective against many types of epileptic seizures (both partial and
generalized seizures). Although the exact mechanism of valproate action
is not clear, its effectiveness as a broad-spectrum anticonvulsant is
usually attributed to a combination of actions at multiple molecular
targets (11, 25). A preponderance of evidence suggests that valproate
potentiates GABAergic neurotransmission by increasing GABA levels in
the brain (11, 26-32). The effect may be attributed both to enhanced
GABA synthesis (stimulation of glutamic acid decarboxylase) and
decreased GABA degradation (inhibition of GABA trans-aminase and
succinic semialdehyde dehydrogenase) (11, 33-35). Correspondingly,
many studies have examined valproate-induced GABA release from
neurons and glia, although vesicular versus non-vesicular
(i.e. transporter-mediated) release mechanisms have not been
definitively addressed. These studies as well as those examining the
effect of valproate on GABA uptake by neurons and/or glial cells have
not provided entirely consistent results; however, the available
evidence favors valproate-induced GABA release from nerve terminals in
selected brain regions (e.g. 31, 32, 36-47; for review see
Refs. 11 and 25). In light of this evidence, it is of interest to
characterize the effect of valproate on the GABA transporters expressed
in an expression system where any putative interaction may be examined
by using sensitive biophysical tools.
Here, we have combined electrophysiological and tracer uptake methods
to examine the effect of valproate on the GABA transporters expressed
in Xenopus laevis oocytes. Our results suggest
that valproate enhances the turnover rate of these transport proteins via an allosteric mechanism. The interaction of valproate with GATs
opens new experimental avenues for probing the mechanism of
Na+/Cl Expression in Xenopus Oocytes--
Stage V-VI X. laevis oocytes were injected with 50 ng of cRNA for human
GAT1 (48), mouse GAT3 (10, 49), mouse GAT4 (49), or rat
Na+/iodide symporter (50). Oocytes were maintained in
Barth's medium (in mM: 88 NaCl, 1 KCl, 0.33 Ca(NO3)2, 0.41 CaCl2, 0.82 MgSO4, 2.4 NaHCO3, 10 HEPES, pH 7.4, 50 µg/ml
gentamicin, 100 µg/ml streptomycin, and 100 units/ml penicillin) at
18 °C for 2-21 days until used in experiments. All of the
experiments were performed at 21 ± 1 °C.
Experimental Solutions--
Unless otherwise indicated,
experiments were performed in a NaCl buffer containing (in
mM): 100 NaCl, 2 KCl, 1 CaCl2, 1 MgCl2, and 10 HEPES, pH 7.4. In Na+-free
solutions, NaCl was isosmotically replaced with choline-Cl. In
Cl Tracer Uptake--
Control and mGAT3-expressing oocytes were
incubated for 30 min in solutions containing 100 µM GABA
and/or various concentrations of valproate in addition to 22 nM [3H]GABA (Amersham Biosciences) or 0.65 µM [3H]valproate (American Radiolabeled
Chemicals, St. Louis, MO). Oocytes were washed and solubilized in 10%
sodium dodecyl sulfate, and oocyte [3H]GABA or
[3H]valproate content was determined in a liquid
scintillation counter (Beckman LS 5000CE). For uptake under voltage
clamp (see Fig. 2A), the membrane potential was held at Electrophysiological Measurements and Data Analysis--
The
two-microelectrode voltage clamp technique was used for the recording
of whole-cell transporter-mediated currents. Oocytes were voltage
clamped by using the Warner Oocyte Clamp (OC-725C, Warner Instrument
Corporation, Hamden, CT). In the recording experimental chamber,
oocytes were initially stabilized in the NaCl buffer and the
composition of the bath was changed as indicated. In all of the
experiments, the reference electrodes were connected to the
experimental oocyte chamber via agar bridges (3% agar in 3 M KCl). For continuous holding current measurements,
currents were low pass-filtered at 1 Hz (LPF 8, Warner Instrument
Corporation) and sampled at 10 Hz (pCLAMP 8.1, Axon Instruments, Union
City, CA).
To examine the effect of valproate on steady-state currents, oocytes
were voltage-clamped at
To examine the carrier-mediated presteady-state current transients, the
pulse protocol consisted of voltage jumps (200-400 ms) from the
holding voltage (
To determine the whole-cell membrane capacitance (see Fig. 3) from a
holding potential of
Presteady-state and steady-state curve fittings were performed by using
either SigmaPlot (SPSS Science; Chicago, IL) or software developed in
this laboratory (using a Marquardt-Levenberg algorithm). Unless
otherwise indicated, results from individual oocytes are shown;
however, each experiment was repeated in at least three oocytes from
different donor frogs. Where sample sizes are indicated (N),
they refer to the number of oocytes in which the experiments were
repeated. Unless otherwise indicated, reported errors represent the
mean obtained from data from several oocytes.
Valproate Enhances mGAT3 Turnover Rate without Altering
Ion/GABA Coupling--
In voltage-clamped (
The large variability in the mGAT3 expression level between oocytes
does not allow a reliable quantitative measure of the enhancement of
GABA uptake relative to the GABA-evoked current. Thus, to obtain a
quantitative comparison between the valproate enhancement of the
GABA-evoked current and GABA uptake, we performed GABA uptake under
voltage clamp in the absence and presence of 5 mM valproate
(Fig. 2). In each cell, the GABA-evoked
current was recorded (100 µM GABA and 22 nM
[3H]GABA with or without 5 mM valproate) and
subsequently, [3H]GABA content in the same oocyte was
determined in a liquid scintillation counter (Fig. 2A). The
net-positive charge trans-located into the cell during the recording
period was obtained from the time integral of the GABA-evoked inward
current. A comparison of the inward charge with GABA uptake in each
cell yielded the charge to GABA ratio per mGAT3 transport cycle (9, 10,
51). The ratio was the same with and without valproate (2.2 ± 0.1 charges/GABA) (Fig. 2B). Therefore, valproate enhancement of
the GABA-evoked current was exactly matched by an enhancement in GABA
uptake. The absence of a change in the ion/GABA coupling ratio in the presence of valproate indicates that valproate did not induce additional ionic conductances.
Valproate enhancement of the GABA-evoked mGAT3 current raises the
possibility that the observed effect may be attributed to reversible
trafficking of mGAT3-containing vesicles to and from the plasma
membrane. Thus, in cells expressing mGAT3, the GABA-evoked inward
current and the valproate enhancement of the inward current were
monitored (Fig. 3A) and
concurrently the whole-cell membrane capacitance was measured (Fig.
3B) (see "Experimental Procedures"). In the cell shown
in Fig. 3, base-line membrane capacitance was 260 nanofarads and
no change was observed in the absence or presence of 5 mM
valproate. A similar observation was made in four additional mGAT3-expressing cells. Thus, valproate does not lead to a significant change in the total surface area of the plasma membrane.
We tested the possibility that valproate may be recognized as a
substrate by mGAT3 and, as such, transported across the plasma membrane. [3H]Valproate uptake was the same in control as
well as mGAT3-expressing oocytes (N Valproate Decreases the Apparent Affinity for GABA--
We
examined the effect of valproate on the steady-state apparent affinity
of mGAT3 for Na+, Cl Valproate Enhancement of the Turnover Rate Is Specific to the GABA
Transporters--
The effect of valproate is common to other GABA
transporter isoforms (Fig. 6). Valproate
enhanced the GABA-evoked inward current mediated by human GAT1 (hGAT1)
and mGAT4 in a similar manner as it did for mGAT3. In general, the
effects on the steady-state as well as the presteady-state (see below)
properties were qualitatively similar to those shown for mouse GAT3. As
valproate has significant membrane permeability (see Fig. 4), we were
concerned that the effect may reflect a nonspecific membrane or
cytoplasmic action of this agent. If that were true, it would be
expected that another Na+-coupled transporter with
mechanistic features similar to those of GABA transporters (9, 51)
would be affected in a similar fashion by valproate. When tested
against the rat Na+/iodide symporter, no valproate
enhancement of the substrate-evoked current was observed (Fig. 6).
Valproate Increases the Rate of Presteady-state Current
Relaxations--
In response to step changes in the membrane voltage,
GABA transporters exhibit presteady-state current transients (Fig.
7) (3, 9, 10, 53-55). These transient
currents are thought to represent conformational changes associated
with partial reactions of the transport cycle (see Fig. 11) and are
composed of fast and slow components (10, 54). In this study, we focus
only on the slow transients of mGAT3 that comprise the major fraction of the voltage-induced charge movements (10). Because of the Cl
Because of the slow return of the charge (OFF transients) in mGAT3
following a voltage pulse (see Fig. 7A and Ref. 10), we
elected to use hGAT1 to more clearly demonstrate the effect of
valproate on the rates of the ON and OFF transitions in response to
voltage pulses (Fig. 8). Valproate (50 mM) led to a
dramatic reduction in the time constants for the relaxation of both the ON and OFF transients (Fig. 8, A-C, E
and F). The maximal charge (Qmax) was
not altered at reduced [Cl Valproate Enhances the Presteady-state Charge Movements at Reduced
Na+ and Cl
At zero [Cl
As GABA leads to a concentration-dependent reduction in
Qmax (10), no charge movements are observed in
the presence of saturating concentrations (1 mM) of GABA
(Fig. 9, compare panels E and F). In the absence
of valproate, the GABA concentration for 50% reduction in
Qmax was 6 ± 1 µM
(n = 4) and that in the presence of valproate (10 mM) was 25 ± 1 µM (n = 3) (Fig. 10C).
We propose that valproate leads to an increase in the turnover
rate of GABA transporters and that the effect involves an important rate-limiting step in the transport cycle. Our data do not allow us to
identify the partial step in the transport cycle that is altered by
valproate; however, we have shown that valproate increases the turnover
rate for the forward mode of the transporter (Fig. 11A) as well as increases
the rates of conformational changes of the empty carrier
(presteady-state relaxations) (Fig. 11B, shaded steps). Valproate interaction with the transporter appears to be
allosteric involving a site other than the GABA binding site, because
valproate is not a transported substrate of GATs and valproate interaction does not compete with GABA. Indeed, valproate enhances GABA
trans-location across the plasma membrane in the forward transport
mode. Despite enhancement of GABA transport across the plasma membrane,
the ion/GABA coupling ratio remains the same, suggesting that the
transport cycle remains tightly coupled. Valproate interaction with the
GABA transporters is specific as other short chain fatty acids such as
butanoic acid and pentanoic acid were without effect (data not shown).
Furthermore, valproate did not enhance the rate of transport for the
Na+/iodide symporter, a Na+-coupled transporter
with mechanistic features similar to those of the GABA transporters.
Examination of transporter presteady-state kinetics suggests that
valproate increases the apparent affinity of the empty transporter for
Na+. The presteady-state charge movements further suggest
that valproate can interact with the transporter in the absence of GABA
and Cl-aminobutyric acid (GABA) transporter (mouse GAT3)
expressed in Xenopus laevis oocytes. In the
absence of GABA, valproate (up to 50 mM) had no noticeable
effect on the steady-state electrogenic properties of mGAT3. In the
presence of GABA, however, valproate enhanced the GABA-evoked
steady-state inward current in a dose-dependent manner with
a half-maximal concentration of 4.6 ± 0.5 mM. Maximal enhancement of the GABA-evoked current was 275 ± 10%.
Qualitatively similar observations were obtained for human GAT1 and
mouse GAT4. The valproate enhancement did not alter the Na+
or Cl
dependence of the steady-state GABA-evoked
currents. Uptake experiments under voltage clamp suggested that the
valproate enhancement of the GABA-evoked current was matched by an
enhancement in GABA uptake. Thus, despite the increase in GABA-evoked
current, ion/GABA co-transport remained tightly coupled. Uptake
experiments indicated that valproate is not transported by mouse
GAT3 in the absence or presence of GABA. Valproate also enhanced
the rate of the partial steps involved in transporter presteady-state
charge movements. We propose that valproate increases the turnover rate
of GABA transporters by an allosteric mechanism. The data suggest that at its therapeutic concentration, valproate may enhance the activity of
neuronal and glial GABA transporters by up to 10%.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
CONCLUSION
REFERENCES
-Aminobutyric acid
(GABA)1 is the most abundant
inhibitory neurotransmitter in the central nervous system. Transport of
GABA into cells is accomplished by
Na+-dependent and Cl
-facilitated
GABA transporters (GATs) found in the plasma membrane of neurons and
glia (1-10). Thus, the GABA transporters regulate synaptic and
extra-synaptic concentrations of GABA and, in this capacity, are partly
responsible for the regulation of inhibitory neurotransmission in the
nervous system. Because of the inhibitory role of GABA, potentiation of
GABAergic neurotransmission via inhibition or reversal of the GABA
transporters is believed to have therapeutic value in treating
epileptic seizures and stroke (11-13). Indeed, inhibitors of the GABA
transporters are known to increase GABA levels in the brain (14-16).
These agents exhibit anticonvulsant activity in animal models, and one
(tiagabine) that preferentially targets the most abundant GABA
transporter isoform in the brain (GAT1) has been in clinical use since
1997 (16-22). Several other clinically used antiepileptic drugs are reported to act, at least in part, via potentiating GABA-mediated inhibition in the brain (11, 23); however, little is known regarding
the potential effect of these drugs on the GABA transporters (24).
/GABA co-transport.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
CONCLUSION
REFERENCES
-free solutions, NaCl, KCl, CaCl2, and
MgCl2 were isosmotically replaced with corresponding
gluconate salts. Choline and gluconate do not interact with the GABA
transporters (3, 9, 10). GABA, sodium-valproate, and/or valproic
acid were added to the above solutions as indicated, and the necessary
pH adjustments were made in solutions containing valproate. In
solutions containing sodium-valproate, the total cation and anion
concentrations were maintained by isosmotic substitution of NaCl with
sodium-valproate and/or sodium-gluconate. For the experiment involving
the Na+/iodide symporter, ClO
60
mV and the oocytes were initially incubated in the NaCl buffer until
base line was established. GABA (100 µM) or GABA (100 µM) and valproate (5 mM) in addition to
[3H]GABA (22 nM) were added to the perfusion
solution for 5-10 min, and the inward current was recorded. At the end
of the incubation period, GABA, valproate, and the isotope were removed
from the perfusion solution until the holding current returned to the
base line. The oocytes were removed from the recording chamber, washed in ice-cold choline-Cl buffer, and solubilized in 10% sodium dodecyl sulfate. The net positive charge trans-located into the cell was obtained from the time integral of the GABA-evoked inward current and
correlated with GABA influx in the same cell (10, 51).
60 mV. Substrate-induced steady-state co-transporter currents were obtained from the difference between the
steady-state currents in the absence and presence of GABA or GABA plus
valproate. The effects of substrate concentration ([GABA]o,
[Na+]o, and [Cl
]o) on the
steady-state kinetics were determined by non-linear curve fitting of
the induced currents (I) to Equation 1,
where S is the substrate ([GABA]o,
[Na+]o, or [Cl
(Eq. 1)
]o),
I
50 mV to a series of test voltages (Vm) from
+80 to
148 mV in 19-mV steps. Currents were low pass-filtered at 500 Hz and sampled at 2 kHz. At each voltage, the steady-state GABA-evoked current was obtained as the difference in steady-state current in the
absence and presence of GABA (or GABA plus valproate).
50 mV) to test voltages ranging from +80 to
150 mV
in 10- or 15-mV steps. Unless otherwise indicated, voltage pulses were
separated by an interval of at least 4 s to allow for complete
relaxation of mGAT3 OFF transients (10). Currents were low
pass-filtered at 1 kHz and sampled at 12.5 kHz without averaging. To
obtain the transporter presteady-state currents, at each
Vm, the total current, I(t),
was fitted to Equation 2,
where t is time;
I1e
(Eq. 2)
t/
1 is the
oocyte-capacitive transient current with initial value
I1 and time constant
1;
I2e
t/
2 is the
transporter transient current with initial value
I2 and time constant
2; and
Iss is the steady-state current (52). At each
Vm, the transporter-mediated charge (Q)
was obtained by time integration of the transporter transient currents.
The charge-voltage (Q-V) relations obtained were then fitted
to a single Boltzmann function as shown in Equation 3,
where Qmax = Qdep
(Eq. 3)
Qhyp (Qdep and
Qhyp are Q at depolarizing and
hyperpolarizing limits, respectively); z is the apparent valence of moveable charge;
is the fraction of the membrane electric field traversed by the charge; V0.5 is
the potential for 50% charge movement; F is Faraday's
constant; R is the gas constant, and T is the
absolute temperature.
60 mV, the membrane was prepulsed to
100 mV
for 500 ms and a 5-mV hyperpolarizing pulse (10 ms) was applied.
Currents were low pass-filtered at 5 kHz and sampled at 10 kHz. After
subtraction of the steady-state currents, the whole-cell capacitance
was determined from the integral of the oocyte-capacitive transients
(Cm = q/Vt, where
Cm is the membrane capacitance, q is the charge
obtained from the integral of the capacitive transient current, and
Vt is the test voltage amplitude (5 mV)) and
reported as the average of values obtained from the ON and OFF
transients. The choice of the prepulse potential (
100 mV) was based
on the mGAT3 Q-V relationship, because at this voltage, no
carrier-mediated charge movement is present (see Fig. 7C)
(10).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
CONCLUSION
REFERENCES
60 mV) oocytes
expressing mGAT3, application of valproate (5 mM) to the
bathing medium had no effect on the holding current (Fig.
1A, left panel).
Valproate concentrations as high as 50 mM did not elicit a
mGAT3-mediated response (data not shown). In the presence of GABA (100 µM), however, the addition of valproate (5 mM) enhanced the GABA-evoked inward current by
100%
(Fig. 1A, right panel). The effect of valproate
was reversible, because after its washout from the bath, the
GABA-evoked current returned to its initial level. Moreover, the effect
of valproate did not diminish on repeated applications. Valproate led
to an enhancement of the GABA-evoked current at all of the voltages tested (
148 to +80 mV) (Fig. 1B). After valproate washout,
the GABA-evoked I-V relation was similar to that obtained
before exposure to valproate (Fig. 1B). Valproate
enhancement of the GABA-evoked inward current was saturable with a
half-maximal concentration of 4.6 ± 0.5 mM
(n = 3) (Fig. 1C). Maximum enhancement was
275 ± 10%. Correspondingly, in non-voltage-clamped oocytes,
valproate enhanced GABA uptake in a concentration-dependent
manner (Fig. 1D). Valproate alone (up to 50 mM)
had no effect on the steady-state electrical properties of
99%
control or mGAT3-expressing oocytes. However, in a small fraction of
cells (
1%), high valproate concentrations (>10 mM) led
to a small outward current of up to
5 nA. These cells
were not used for further investigation.
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Fig. 1.
Enhancement of mGAT3 GABA transport by
valproate. A, current traces from a mGAT3-expressing
oocyte maintained at a holding potential of 60 mV. GABA and/or
valproate were added as indicated by the bars. In the
absence of GABA, valproate alone (5 mM) had no noticeable
effect on the holding current of mGAT3-expressing cells (left
panel). A similar observation was made at valproate concentrations
of up to 50 mM. In the presence of GABA (100 µM), valproate (5 mM) enhanced the
GABA-evoked response by
100% (right panel). The effect
of valproate was reversible with no apparent sensitization or
desensitization on repeated applications. Previous applications of
valproate had no effect on subsequent GABA responses. Thus, the
valproate effect was seen only when GABA was present. B,
current-voltage (I-V) relations of the mGAT3 GABA-evoked
(100 µM) current were obtained before exposure to
valproate (filled circles), in the presence of 10 mM valproate (filled squares), and after
exposure to valproate (open circles). Similar I-V
curves were obtained in six mGAT3-expressing cells. C,
valproate enhancement of the GABA-evoked inward current
(I
60 mV). Inset shows the valproate
effect within its therapeutic concentration range in the plasma and
cerebrospinal fluid. D, valproate also enhanced GABA uptake
into mGAT3-expressing oocytes in a concentration-dependent
manner (N
15 cells at each valproate concentration).
The incubation medium contained 100 µM GABA, 22 nM [3H]GABA, and valproate at the indicated
concentration. Values were normalized with respect to that in the
absence of valproate (6.42 ± 0.38 pmol/min/oocyte). In control
oocytes from the same batch, GABA uptake was not altered by valproate
(0.29 ± 0.01 pmol/min/oocyte; N
10; data not
shown). Similar results were obtained in four batches of cells.
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Fig. 2.
Tight ion/GABA coupling is maintained despite
valproate-enhanced GABA transport. A, current trace
from an oocyte expressing mGAT3 and maintained at 60 mV. Base line
was established in the NaCl buffer, and at the time indicated by the
bar, GABA (100 µM) and [3H]GABA
were perfused into the chamber. After washout of GABA and the isotope
and return to the base line, the oocyte was washed in ice-cold
choline-Cl buffer and counted in a liquid scintillation counter. The
time integral of the GABA-evoked current trace yielded the total
net-positive charge transported into the cell during the recording
period. In an additional group of cells, valproate (5 mM)
was also added to the perfusion solution. In control cells, GABA did
not induce an inward current (data not shown). B, the ratio
of net inward charge (obtained from the integral of the inward current)
to GABA uptake (obtained from [3H]GABA influx) determined
in the same cells was 2.2 ± 0.1 charges/GABA in the absence
(open circles; n = 7) and presence
(filled circles; n = 8) of valproate. This
experiment was repeated in two other batches of oocytes, and the
results were identical to those shown here.
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Fig. 3.
Valproate does not alter vesicle trafficking
to the plasma membrane. Panel A shows a current trace
from an oocyte expressing mGAT3 (Vm = 60 mV). At
the time indicated by the bars, GABA (100 µM)
and valproate (5 mM) were added to the perfusion chamber
and the inward current (A) and the membrane capacitance
(B) were monitored concurrently. To measure the whole-cell
capacitance, every 5 s, the membrane was prepulsed to
100 mV for
500 ms, and a 5-mV hyperpolarizing pulse (10 ms) was applied.
Whole-cell capacitance was obtained as the average of values determined
for the ON and OFF capacitive transients (see "Experimental
Procedures"). This protocol led to periodic gaps (
1 s in duration)
in the current trace of panel A, which are too short in
duration to be noticed at the time scale shown. Despite a large
enhancement of the GABA-evoked inward current, the whole-cell
capacitance (260 nanofarads) remained unchanged, suggesting unaltered
vesicle trafficking to and/or from the plasma membrane (see
"Discussion"). A similar observation was obtained in five
mGAT3-expressing cells.
20) (Fig.
4). Moreover, [3H]valproate
uptake was not altered in the presence of GABA (100 µM)
(N
20). Thus, valproate is not a transported
substrate of mGAT3.
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Fig. 4.
Valproate is not a transported substrate of
mGAT3. Uptake of [3H]valproate (0.65 µM; total valproate concentration, 2 mM) was
examined in control and mGAT3-expressing oocytes in the absence and
presence of 100 µM GABA. The experiments were performed
in the NaCl buffer. Neither mGAT3 expression nor GABA (100 µM) had an effect on valproate uptake by control or
mGAT3-expressing cells. Data represent the mean ± S.D.
(N 20). In the same batch of oocytes, GABA uptake
was 0.45 ± 0.02 pmol/min/oocyte (n = 10) in
control cells and 12.23 ± 3.10 pmol/min/oocyte (n = 17) in mGAT3-expressing cells (data not shown). This experiment was
repeated with oocytes from four different batches with similar results.
Therefore, valproate is not transported by mGAT3. The relatively high
uptake rate of valproate in control cells as well as in
mGAT3-expressing cells is most probably because of the moderate
oil-water partition coefficient of the protonated (uncharged) form of
valproate (valproic acid). As the experiments were carried out at pH
7.4, it is unlikely that the observed transport rates would result from
the activity of the endogenous H+-driven monocarboxylate
transporter.
, and GABA (Fig.
5). In these experiments, substrate
(Na+, Cl
, or GABA) kinetics was examined in
the absence and presence of valproate in the same cells, while the
concentrations of the other two co-substrates were held constant.
Valproate enhancement of the evoked current was
Na+-dependent, because in the absence of
Na+, no GABA-evoked current was observed in the absence or
presence of valproate (Fig. 5A). Valproate (5 mM) had no effect on the half-maximal concentration
(K
and 100 µM GABA) (n = 3) (Fig. 5A).
Similarly, there was no significant effect on the Na+ Hill
coefficient (2.0 ± 0.1 versus 2.3 ± 0.1;
n = 3). The half-maximal concentration for
Cl
enhancement (K
]o (
150% at all
Cl
concentrations) (Fig. 5B). The half-maximal
concentration for GABA activation of the currents
(K
)
(n = 3) (Fig. 5C).
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Fig. 5.
Valproate decreases the apparent affinity for
GABA. In oocytes expressing mGAT3, steady-state inward currents
were activated in the absence and presence of valproate at increasing
concentrations of Na+, Cl , or GABA while the
concentrations of the other two co-substrates were held constant
(Vm =
60 mV). A, in the absence of
Na+, valproate and GABA did not induce an inward current.
Valproate had no effect on the apparent affinity of mGAT3 for
Na+. The apparent affinity constant for Na+ was
15 ± 2 mM in the absence (open circles)
and 13 ± 2 mM in the presence of 5 mM
valproate (filled circles) (n = 3). The Hill
coefficient was 2.0 ± 0.1 in the absence and 2.3 ± 0.1 in
the presence of valproate (n = 3).
[Cl
]o was 101 mM, and [GABA] was
100 µM. B, valproate did not alter the
apparent affinity for Cl
. The apparent affinity constant
for Cl
was 0.4 ± 0.1 mM in the absence
(open circles) and 0.4 ± 0.1 mM in the
presence of 10 mM valproate (filled circles)
(n = 3). [Na+]o was 100 mM, and [GABA] was 100 µM. The magnitude of
valproate enhancement was approximately the same at all
Cl
concentrations. C, valproate (10 mM) led to a decrease in the apparent affinity of mGAT3 for
GABA. The apparent affinity constant for GABA was 8 ± 1 µM in the absence (open circles) and 20 ± 1 µM in the presence of 10 mM valproate
(filled circles) (n = 3).
[Na+]o was 100 mM, and
[Cl
]o was 96 mM. The results shown
in each panel were obtained from the same oocyte.
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Fig. 6.
Valproate enhancement of the turnover rate is
specific to the GABA transporters. Representative recordings are
shown for oocytes expressing hGAT1 (left panel), mGAT4
(middle panel), and rat Na+/iodide symporter
(rNIS) (right panel). Oocytes were
voltage-clamped at 60 mV, and at the time indicated by the
bars, the appropriate transporter substrate (GABA or
ClO
dependence of the presteady-state currents (3, 9, 10, 53), in these studies, valproate isosmotically replaced gluconate while
the Cl
concentration was maintained at the values
indicated. The most pronounced effect of valproate was an increase in
the rate of relaxation of the transients (Fig. 7). In the GABA
transporters, the relaxation of the ON transients is
voltage-dependent. The time constant of the ON relaxation
plotted as a function of the test voltage follows a bell-shaped
distribution (see Fig. 8E for hGAT1 data) (10, 53, 54). For the OFF response, the relaxation time
constants are only weakly voltage-dependent (see Fig.
8F for hGAT1 data). For the ON transients of mGAT3, the peak
relaxation time constant was 36 ± 4 ms (at
5 mV) in the absence
of valproate and 25 ± 3 ms (at
25 mV) in the presence of
valproate (n = 6). The effect of valproate was more
pronounced on the mGAT3 OFF transients (Fig. 7A and
B, compare OFF transients; see also Fig. 8). We have shown
previously that mGAT3 OFF transients relax slowly to a steady state
with a time constant of
1 s (10). As evident in Fig. 7, panel
B, the OFF transients reached steady state more rapidly in the
presence of valproate (
OFF was 21 ± 5 ms;
n = 6) (Fig. 7B) than in its absence (Fig.
7A) (see below for further details). Valproate led to a
shift of the charge-voltage (Q-V) relationship to more
negative membrane potentials (Fig. 7C).
V0.5 of the Q-V curve shifted by
21 ± 3 mV (n = 6) in the presence of saturating concentrations of valproate (50 mM). The valproate
concentration for half-maximal shift in the V0.5
was 5.9 ± 0.5 mM (n = 6) (Fig. 7D). Valproate had no effect on the maximum charge
(Qmax) moved in response to the voltage pulses
(Fig. 7, C and E). Finally, the apparent valence
of the moveable charge (z
) was not altered by valproate
(Fig. 7F).
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Fig. 7.
Valproate does not alter the maximum
presteady-state charge movement (Qmax) at
saturating Na+ and Cl concentrations.
A and B, current traces were recorded from a
mGAT3-expressing oocyte in response to a series of 200-ms voltage
pulses ranging from +80 to
120 mV in 10-mV steps. The major cation
and anion composition of the bath was as follows: A,
[Na+]o = 100 mM,
[Cl
]o = 56 mM, and [gluconate] = 50 mM. B, [Na+]o = 100 mM, [Cl
]o = 56 mM, and
[valproate] = 50 mM. Notice the effect of valproate on
the OFF transients. C, valproate led to a shift of the
charge-voltage (Q-V) relationship toward negative membrane
potentials. V0.5 was 29 mV in the absence and 9 mV in the presence of valproate (10 mM). D, the
shift in V0.5 was
concentration-dependent with a half-maximal concentration
of 5.9 ± 0.5 mM (n = 6). The maximal
shift in V0.5 was
21 ± 3 mV
(n = 6). E and F, valproate had
no effect on the maximal charge (Qmax) or the
apparent valence of the moveable charge (z
).
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Fig. 8.
Valproate increases the rate of
presteady-state current relaxations. A-C,
current traces were recorded from a human GAT1-expressing oocyte in
response to a series of 400-ms voltage pulses ranging from +60 to 150
mV in 15-mV steps. The major cation and anion composition of the bath
was as follows: A, [Na+]o = 100 mM and [Cl
]o = 106 mM. B, [Na+]o = 100 mM, [Cl
]o = 56 mM, and
[gluconate] = 50 mM. C,
[Na+]o = 100 mM,
[Cl
]o = 56 mM, and [valproate] = 50 mM. Notice that valproate speeds up the relaxation of
the ON and OFF presteady-state current transients. Records shown in
panels A-C were obtained from the same cell.
D, charge-voltage (Q-V) relationships for the
records shown in panels A-C. The maximal charge
(Qmax) was not altered at reduced
[Cl
]o, and in addition, valproate had no effect
on Qmax. The mid-point of the Q-V
curve (V0.5) shifted to the left as
[Cl
]o was reduced from 106 to 56 mM. V0.5 shifted from
29 to
47
mV. The addition of 50 mM valproate, however, shifted the
Q-V relationship toward positive membrane potentials.
V0.5 shifted from
47 to
29 mV. The apparent
valence of the moveable charge (z
) was not altered by the
reduction in [Cl
]o or by addition of valproate
(data not shown). E, similar to the Q-V
relationships, the
ON-V curves shifted to the
left as [Cl
]o was reduced and again shifted to
the right as valproate was added. Valproate led to a
2.5-fold
reduction in the ON relaxation time constants. At its maximum value (at
48 mV),
ON was 99 ms without and 40 ms (at
29 mV)
with valproate. F, valproate also led to a
2.5-fold
reduction in the OFF relaxation time constants. (90 versus
36 ms).
]o (3, 9), and in
addition, similar to the observation made with mGAT3, valproate (up to
50 mM) had no effect on Qmax (Fig.
8D). In contrast to the observed response of mGAT3,
valproate led to a right shift of hGAT1 Q-V relationship.
The maximum shift in V0.5 was 18 ± 2 mV
(n = 4). The mid-point of the Q-V curve (V0.5) shifted to the left as
[Cl
]o was reduced from 106 to 56 mM
(gluconate replacement). V0.5 shifted from
29 ± 4 to
52 ± 4 mV (n = 4). However,
the addition of 50 mM valproate (equimolar replacement of
gluconate) shifted the Q-V relationship toward positive
membrane potentials. V0.5 shifted from
52 ± 4 to
33 ± 4 mV (n = 4). The apparent valence
of the moveable charge (z
) was not significantly altered by the reduction in [Cl
]o or by addition of
valproate (1.0 ± 0.1 versus 1.2 ± 0.1). Similar
to the Q-V relationship, the
ON-V
curve shifted to the left as [Cl
]o was reduced
and again shifted to the right as valproate was added. Valproate led to
a
2.5-fold reduction in the ON relaxation time constants. At its
maximum value (at
52 mV)
ON was 104 ± 4 ms
without valproate and 42 ± 3 ms (at
33 mV) with valproate (n = 4). Valproate also led to a
2.5-fold reduction
in the OFF relaxation time constants (101 ± 3 ms
versus 37 ± 2 ms (n = 4)).
Concentrations--
The Na+,
Cl
, and GABA dependence of mGAT3 presteady-state charge
movements were examined in the absence and presence of valproate in the
same cells (Figs. 9 and 10). We have
shown previously that the charge movements of mGAT3 strictly depend on
external Na+, because at zero [Na+]o,
no mGAT3-mediated charge movement is detected (10). In the absence of
external Na+, no charge movement was detected in the
absence or presence of valproate (Fig. 10A). However,
significantly more charge was moved at lower
[Na+]o in the presence of valproate than in its
absence (Figs. 9, A and B, and 10A).
At 10 mM [Na+]o,
Qmax was 8 nanocoulombs in the absence
(Fig. 9A) and 15 nC in the presence of 10 mM
valproate (Fig. 9B). The increase in
Qmax at low [Na+]o
occurred despite the fact that at each concentration, the
Q-V relationship was shifted toward more negative potentials (by
17 mV) in the presence of valproate (see Fig. 7D). At
saturating [Na+]o, Qmax
was the same with or without valproate (Fig. 10A). Valproate
led to an apparent decrease in the half-maximal concentration for
Na+ activation of the presteady-state charge movements
(Fig. 10A). K
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Fig. 9.
Valproate increases the presteady-state
charge movements at reduced Na+ and Cl
concentrations. Current traces were recorded from a
mGAT3-expressing oocyte in response to voltage pulses (200 ms) ranging
from +80 to
120 mV in 10-mV steps. A and B, at
10 mM [Na+]o, significantly more
charge was seen in the presence of 10 mM valproate
(B) than in its absence (A).
Qmax was 8 nC without and 15 nC with valproate.
Na+ was isosmotically replaced with choline. C
and D, in the nominal absence of external Cl
,
more charge was observed in the presence of 10 mM valproate
(D) than in its absence (C).
Qmax was 7 nC without and 17 nC with valproate.
E and F, saturating concentrations of GABA lead
to the elimination of charge movements. The relevant composition of the
bathing medium is indicated (in mM) for each panel. Records
from all six panels were obtained from the same cell. The scale
bars shown apply to all panels.
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Fig. 10.
Effect of valproate on Na+,
Cl , and GABA dependence of the presteady-state charge
movements. Na+, Cl
, and GABA dependence
of mGAT3 presteady-state charge movements were examined in the absence
and presence of 10 mM valproate. Voltage pulses were
similar to those indicated for Fig. 9. A, at nominal zero
[Na+]o, Qmax is below the
resolution of the measurements (
2 nC). In the absence of external
Na+, valproate (10 mM) does not induce
presteady-state charge movements. The half-maximal concentration for
Na+ activation of the charge movements was 43 ± 2 mM (n = 7) in the absence of valproate
(open circles) and 22 ± 4 mM
(n = 7) in the presence of 10 mM valproate
(filled circles). The Hill coefficient was 2.1 ± 0.2 in the absence and 1.1 ± 0.2 in the presence of valproate
(n = 7). For the experiment shown,
Qmax was 18 nC. B, valproate (10 mM) had no effect on the half-maximal concentration for
Cl
enhancement of the charge movements (16 ± 2 mM versus 15 ± 3 mM;
n = 3). For the experiment shown,
Qmax was 35 nC. C, saturating GABA
concentrations lead to the elimination of presteady-state charge
movements (see Fig. 9, E and F). The GABA
concentration for 50% reduction in Qmax was
6 ± 1 µM (n = 4) in the absence of
valproate (open circles) and 25 ± 1 µM
(n = 3) in the presence of 10 mM valproate
(filled circles). For the experiment shown,
Qmax was 28 nC. The results shown in each panel
were obtained from the same oocyte.
]o, the magnitude of mGAT3 charge
movement is reduced by
80% (Fig. 9, compare panels C and
E; see also Fig. 10B) (10). At low
[Cl
]o, more charge was present in the presence
of valproate than in its absence (Fig. 10B). At nominal zero
[Cl
]o, valproate (10 mM) increased
Qmax from 7 to 17 nC (Fig. 9, compare
panels C and D). At nominal zero
[Cl
]o, Qmax was 19 ± 1% of that at 106 mM [Cl
]o in
the absence of valproate and 51 ± 4% in the presence of
valproate (n = 3) (Fig. 10B). The
half-maximal concentration for Cl
enhancement of the
charge movements, however, was not altered by valproate (16 ± 2 mM versus 15 ± 3 mM;
n = 3). At saturating [Cl
]o,
Qmax was the same with or without valproate
(Fig. 10B).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
CONCLUSION
REFERENCES
. However, it cannot be determined whether
valproate can interact with the transporter in the absence of
Na+, because no charge movements are induced by valproate
in the absence of external Na+. Finally, valproate-induced
enhancement is rapid and limited only by the speed of the perfusion
system, and moreover, the enhancement is fully reversible.
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Fig. 11.
Proposed scheme of the effect of valproate
on the GABA transporters. The transport cycle may be schematized
by a series of partial reactions involving binding/dissociation of
ligands at the external or internal membrane surfaces as well as the
translocation of the ligand-bound or empty binding sites from one
membrane surface to another. C, carrier; Na,
Na+; Cl, Cl ; and G,
GABA. The subscripts o and i refer to the outward
and inward, respectively, facing carrier binding sites. A,
valproate enhances the overall rate for the forward transport cycle
(clockwise transitions; i.e.
Na+/Cl
/GABA co-transport into the cell). See
Figs. 1, 2, 5, and 6 for supporting evidence. B, the charge
movements occur in response to voltage perturbations and represent
Na+ and Cl
binding/dissociation as well as
conformational changes of the empty transporter. The shaded
steps correspond to those thought to be responsible for the
presteady-state charge movements. Valproate decreases the time
constants for the ON and OFF relaxation of the presteady-state charge
movements. Thus, the rates for both the ON transitions
(counterclockwise) and the OFF transitions
(clockwise) are increased by valproate. See Figs. 7 and 8
for supporting evidence.
At least three observations suggest that valproate interaction with mGAT3 is not at the GABA binding site. (i) Valproate alone neither evokes an inward current nor is it itself transported across the plasma membrane alone or in the presence of GABA. (ii) Increasing concentrations of valproate enhance both the GABA-evoked current and GABA uptake, whereas interaction at the GABA binding site would be expected to lead to competitive inhibition of GABA uptake. (iii) Valproate had no effect on the maximal charge moved in response to voltage pulses (Qmax). Qmax is a measure of the total number of functional transporters available to bind substrate (56). As transported substrates lead to a concentration-dependent reduction in Qmax (Fig. 10C) (10, 51), the results suggest that interaction of valproate with the GABA transporters occurs in an entirely different fashion from that of a transported substrate. Because of the moderate membrane permeability of valproate, it is not possible to know whether this drug acts at the extracellular, intracellular, or membrane-spanning region of the transporter.
The effect of valproate cannot be attributed to an increase in the
total number of transporters caused by vesicle trafficking to the
plasma membrane. Four observations support this view. (i) The total
number of transporters in the plasma membrane (as determined from
Qmax) was not changed by valproate. (ii) The
effect was fully reversible and repeatable in the same cell, persisting
for as long as the experiment was continued. It seems unlikely that
such mGAT3-containing vesicle insertion could be balanced rapidly and precisely by recruitment of mGAT3 in retrieved vesicles. (iii) The
effect was specific to the GABA transporters as the transport rate for
the Na+/iodide symporter was not altered. (iv) There was no
change in the whole-cell capacitance in the presence of valproate. This is shown clearly in Fig. 3, A and B. We have
shown previously that heterologous membrane proteins expressed in
oocytes are targeted to the plasma membrane in 100-nm diameter vesicles
containing 5-40 copies of the expressed protein (57). In the oocyte of Fig. 3A, the GABA-evoked current was enhanced by 50 nA.
If it is assumed that the enhancement was because of insertion of new mGAT3 copies in the plasma membrane, it can be estimated that a total
of
7 × 1010 transporters were newly inserted into
the membrane (I = NRze, where I is current,
N is the number of transporters, R is the turnover rate (2 s
1 at
60 mV; see Ref. 10),
z is the net charge trans-located across the membrane per
transport cycle (2.2; see legend to Fig. 2B), and
e is the elementary charge). Assuming that there were 5-40
copies of mGAT3 per vesicle, 2 × 109 to 14 × 109 vesicles would have been expected to fuse with the
plasma membrane, leading to a 2-16-fold increase in the total surface
area of the oocyte. Clearly, with a resolution of
10 nanofarads,
such an increase in capacitance (260 nanofarads to at least 520 nanofarads) would have been detected (for example see Ref. 58).
A prominent effect of valproate was an increase in the rate of the
presteady-state ON and OFF relaxations. Upon application of
depolarizing voltage pulses, the ON transients represent the release of
Na+ and Cl followed by reorientation of the
empty carrier (Fig. 11B, shaded steps). Upon
return from the test voltage to the holding voltage, the OFF transients
represent the return of the binding sites to the external medium,
Na+ and Cl
entry into the membrane electric
field, binding, and subsequent ligand-induced conformational changes
(Fig. 11B) (3, 52-55). As presteady-state charge movements
are obtained in the absence of GABA, the data suggest that valproate
also increases the rates associated with voltage-induced conformational
changes of the empty carrier.
Valproate also led to a small reduction in the apparent affinity for
GABA. Both the half-maximal concentration for steady-state GABA-evoked
inward current and the GABA concentration for 50% reduction in
Qmax were increased by valproate. The values
increased from 6 µM in the absence of valproate to
20 µM in the presence of 10 mM valproate.
Apparently, valproate reversibly transforms mGAT3 into a lower
affinity, higher capacity transporter. Interestingly, valproate was
also reported to decrease the GABA affinity of the astroglial GABA
uptake system (44), although maximal transport rates were not altered.
At saturating Na+ and Cl concentrations,
valproate did not alter the maximal charge moved in response to voltage
pulses (see Figs. 7, 8, and 10), suggesting that valproate entry/exit
into/out of the membrane electric field does not contribute to
presteady-state charge movements. Valproate did not alter the apparent
valence of the moveable charge (z
), suggesting that the
same number of charges moved the same distance within the membrane
electric field in the absence or presence of valproate. Valproate led
to a decrease in the half-maximal concentration for Na+
activation (43 versus 22 mM) of the charge
movements but had no effect on the half-maximal concentrations for
Cl
enhancement (15 mM) of the charge
movements. Therefore, the results suggest that valproate increases the
apparent affinity of the empty transporter for Na+. Thus,
by increasing the affinity of the empty carrier for Na+,
significantly more charge can be moved at lower Na+ and
Cl
concentrations. This effect may contribute to the
enhancement of transporter turnover rate induced by valproate. In
contrast, valproate does not alter the apparent Na+
affinity of the GABA-loaded transporter (see Fig. 5A). As
the presteady-state transitions (Fig. 11B, shaded
steps) represent only a subset of those of the entire transport
cycle (Fig. 11A), the data suggest that the effect of
valproate is complex and may also involve partial steps other than
those responsible for the voltage-induced presteady-state transitions.
The data do not allow us to specify the steps altered by valproate.
Our results are in contrast to those obtained by Eckstein-Ludwig et al. (24) who report the inhibition of mouse GAT1-mediated GABA uptake by valproate, although it was also reported that valproate did not alter the steady-state or presteady-state currents of mouse GAT1. The authors propose a valproate-induced dissociation of GABA uptake and GABA-induced currents. We were unable to observe such effects under our experimental conditions. Indeed, we have provided direct demonstration of tight coupling between GABA uptake and steady-state GABA-induced currents in the absence and presence of valproate. Differences in the GAT isoform and/or valproate concentration used may be responsible for the observed discrepancy between our results and those of Eckstein-Ludwig et al. (24).
In humans, the therapeutic concentration of valproate in the plasma
ranges from 0.28 to 0.69 mM (11). Because of valproate breakdown to metabolic byproducts as well as carrier-mediated efflux
from the brain, valproate concentration in the cerebrospinal fluid is a
fraction of that in plasma and ranges from 0.042 to 0.19 mM
(11). The half-maximal concentration for valproate enhancement of the
GAT turnover rate is 4.5 mM, and maximal enhancement is
275%. Therefore, we predict that at therapeutic levels of
valproate, the activity of the GABA transporters may be enhanced by up
to 10%. Valproate has been shown to lead to a 15-40% elevation of GABA levels in the cerebrospinal fluid (25). Although our data do not
allow us to speculate on the role played by the GABA transporters, at
least in principle, it is possible that valproate enhancement of the
GABA transporters may contribute to the observed elevation of GABA
levels in the cerebrospinal fluid. By stimulating GABA synthesis and
inhibiting GABA degradation, valproate increases the nerve ending GABA
pool (36, 41), which may favor GABA release via the reversal of the
GABA transporters (8). Therefore, valproate enhancement of the GABA
transporter turnover rate may add to its effectiveness in facilitating
GABA release from cells, leading to GABA potentiation in the brain.
These findings add support to the notion that the effectiveness of
valproate as an anticonvulsant results from its combined action at
multiple molecular targets in the brain.
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CONCLUSION |
---|
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---|
Since its fortuitous discovery as an anticonvulsant in 1962, valproate has become one of the most widely used drugs of its class, perhaps because of its wide spectrum of
anticonvulsant activity against different types of seizures. The wide
spectrum of activity most certainly arises from diverse molecular
actions. Here, we have presented data for an additional role of
valproate in the central nervous system, the enhancement of the
turnover rate of the GABA transporters. We predict that at therapeutic concentrations, valproate may enhance the activity of neuronal and
glial GABA transporters by up to 10%.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Gail M. Drus, Michael J. Errico, William Lee, and Erik B. Malarkey for technical assistance and Dr. Nancy Carrasco for the gift of the plasmid containing the rat Na+/iodide symporter.
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FOOTNOTES |
---|
* This work was supported in part by National Institutes of Health Grant S06 GM53933 (to S. E.) and by grants from the Israel Science Foundation and the United States-Israel Binational Scientific Foundation (to N. N.).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: Biological
Sciences Department, California State Polytechnic University, 3801 W. Temple Ave., Pomona, CA 91768-4032. Tel.: 909-869-4182; Fax: 909-869-4078; E-mail: seskandari@csupomona.edu.
Published, JBC Papers in Press, February 20, 2003, DOI 10.1074/jbc.M207582200
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ABBREVIATIONS |
---|
The abbreviations used are:
GABA, -aminobutyric acid;
GAT, GABA transporter;
mGAT, mouse GAT;
hGAT, human GAT;
I-V, current-voltage;
Q-V, charge-voltage;
Qmax, maximum
transporter-mediated charge.
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REFERENCES |
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