The excitatory amino acid transporter EAAT4 is
expressed predominantly in Purkinje neurons in the rat cerebellum
(1-3), and it participates in postsynaptic reuptake of glutamate
released at the climbing fiber synapse (4). Transporter-mediated
currents in Purkinje neurons are increased more than 3-fold by
arachidonic acid, a second messenger that is liberated following
depolarization-induced Ca2+ activation of
phospholipase A2 (5). In this study we demonstrate that
application of arachidonic acid to oocytes expressing rat EAAT4
increased glutamate-induced currents to a similar extent. However,
arachidonic acid did not cause an increase in the rate of glutamate
transport or in the chloride current associated with glutamate
transport but rather activated a proton-selective conductance. These
data reveal a novel action of arachidonate on a glutamate transporter
and suggest a mechanism by which synaptic activity may decrease
intracellular pH in neurons where this transporter is localized.
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INTRODUCTION |
Glutamate transporters play critical roles in synaptic
transmission and in maintaining glutamate homeostasis in the brain (6).
They are encoded by genes belonging to a family of acidic and neutral
amino acid transporters (7), and they exhibit specific localization
patterns. Glutamate transporters found on glia include EAAT1
(excitatory amino acid
transporter 1)/GLAST and EAAT2/Glt-1 (8, 9),
and transporters found on neurons include the widely expressed
EAAT3/EAAC1 (8), the cerebellar-specific EAAT4 (1-3), and the retinal
EAAT5 (10).
Arachidonate is released following activation of postsynaptic glutamate
receptors (11). In synaptosomal preparations, arachidonate inhibits
glutamate uptake (12-14). However, it exerts differential effects on
cloned glutamate transporter subtypes, enhancing EAAT2 and inhibiting
EAAT1 transport (15). Arachidonate inhibits uptake in salamander
retinal glial cells (16). These cells predominantly express an EAAT1
homolog (sEAAT1) that is similarly inhibited by arachidonate when it is
exogenously expressed in oocytes (10, 15). Because arachidonate is
released during synaptic activity and can modulate synaptic
transmission (17, 18), understanding its effects on various glutamate
transporter subtypes is important. Recently, Kataoka et al.
reported an activity-dependent enhancement of glutamate
transporter currents in rat cerebellar Purkinje neurons that was
mediated by arachidonate (5). The present study was designed to examine
the mechanism of the effects of arachidonate on the cloned rat EAAT4
transporter, which is expressed at high levels in Purkinje neurons.
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EXPERIMENTAL PROCEDURES |
The rat EAAT4
cDNA1 was subcloned into
pOG, a vector derived from pBSTA (19) that contains a multiple cloning
site between flanking Xenopus
-globin 5'- and
3'-untranslated sequences. Capped mRNA was transcribed using T7
polymerase and injected into stage V or VI oocytes (approximately 50 ng/oocyte). Recordings and radiolabel uptake assays were made 4-7 days
later as described (15). Extracellular Ringer's solution contained (in
mM) 100 NaCl, 2 KCl, 1.8 CaCl2, 1 MgCl2. Buffers were present at 5 mM and
consisted of MES2/HEPES (pH
6.5), Na/HEPES (pH 7.5), or HEPES/Tris (pH 8.5). Solutions containing
indicated ion substitutions were changed by bath exchange. Recordings
were made using a two-microelectrode voltage clamp circuit (20), and
records were analyzed using pCLAMP 6.0 software (Axon Instruments).
[3H]L-glutamate (1 Ci/mmol; Amersham
Pharmacia Biotech) uptake assays were performed at 25 °C. Following
a 5-min incubation in the indicated concentration of
[3H]L-glutamate (10 µCi/ml), oocytes were
rapidly washed three times in cold Ringer and lysed in 1% SDS, and
scintillation spectroscopy was performed. Arachidonic acid (Calbiochem)
was stored at
20 °C in 100 mM stock solutions in
Me2SO and dissolved in recording solution by sonication
immediately prior to use. All other compounds were from Sigma.
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RESULTS |
Three to four days following injection of RNA transcribed from the
rat excitatory amino acid transporter EAAT4 cDNA Xenopus oocytes displayed >30-fold increased uptake of 1 µM
[3H]L-glutamate. In oocytes voltage-clamped
at
60 mV, EAAT4 currents induced by application of 30 µM L-glutamate were increased upon co-application of 100 µM arachidonate (Fig.
1A). This effect was reversible, although its onset and offset were slower than the solution
exchange times as monitored by the glutamate response (Fig.
1A). Application of 100 µM arachidonate alone
in oocytes expressing rEAAT4 resulted in a small but significant inward
current (Fig. 1B;
2.2 nA ± 0.6, n = 4). This inward current was not observed in uninjected oocytes (1.1 nA ± 0.9, n = 9). At
60 mV, arachidonate increased the magnitude of the steady-state current induced by 30 µM glutamate to 324 ± 41% of its control value
(n = 11). Currents elicited by glutamate in the
presence and absence of arachidonate at a series of membrane potentials
showed that arachidonate enhanced the current amplitude to a greater
extent at more negative potentials (Fig. 1, C and
D; also see Fig. 4). These results are consistent with a
study on Purkinje neuron transporter currents (5) and a recent report
on the human EAAT4 transporter (21).

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Fig. 1.
Arachidonic acid enhances the magnitude of
glutamate-induced currents recorded in voltage-clamped oocytes
expressing rat EAAT4. A, representative cell clamped at
60 mV; compounds were superfused for the times indicated by the
open (30 µM L-glutamate) and
closed bars (100 µM arachidonic acid).
B, application of arachidonate alone induced a current much
smaller than observed with co-application of L-Glu.
C, subtracted (30 µM glutamate-control)
currents recorded during 90-ms voltage jumps between 120 mV and +70
mV. D, currents recorded in the same cell as C
with 100 µM arachidonic acid present. The dashed
line indicates zero current; capacitive artifacts have been
removed for clarity. Holding potential, 70 mV.
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The arachidonate concentration dependence in the presence of a
saturating concentration of glutamate (30 µM) revealed
that the arachidonate effect on the current was saturable, with an EC50 of 135 ± 21 µM (n = 3; Fig. 2A). In the presence
of arachidonate, the apparent affinity of the transporter for glutamate
was unaffected; the EC50 of the current was 1.5 ± 0.2 and 1.5 ± 0.3 µM in control and with 100 µM arachidonate, respectively (n = 6).
The effects of arachidonate on the transport current seemed to be
direct rather than through a metabolite, because coapplication of the
cyclooxygenase inhibitor indomethacin (100 µM) together
with the lipoxygenase inhibitor nordihydroguaretic acid (50 µM) had no effect on the potentiation induced by
arachidonic acid (96 ± 2% of control enhancement, n = 3). To examine whether arachidonate affected the
transport of L-glutamate in oocytes expressing rEAAT4,
uptake of 1 µM or 30 µM
[3H]L-glutamate was assayed in the presence
or absence of 300 µM arachidonate (Fig. 2, C
and D). In marked contrast to its effects on the currents,
arachidonate had no significant effect on the uptake of
L-glutamate into oocytes. Uptake of 1 µM
[3H]L-glutamate was 524 ± 152 fmol/min
in control conditions and 468 ± 86 fmol/min in the presence of
300 µM arachidonate (n = 6, p = 0.74). Uptake of 30 µM
L-glutamate uptake was also not significantly changed by
300 µM arachidonate (1753 ± 506 fmol/min and
1477 ± 271 fmol/min in control and arachidonate, respectively; n = 5; p = 0.64). Arachidonate also had
no effect on L-glutamate uptake in uninjected oocytes (Fig.
2, C and D).

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Fig. 2.
Arachidonate dose-dependently
increases the transport current without increasing glutamate flux.
A, arachidonate concentration dependence of transporter
currents activated by 30 µM glutamate (normalized to the
maximal current at Vm= 70 mV). Points
(mean ± S.E., n = 3) are fitted to the
Michaelis-Menten equation with a K0.5 of 135 µM. B, L-glutamate concentration
dependence of currents in the presence and absence of 100 µM arachidonate. Currents (mean ± S.E.,
n = 6) were normalized to the maximum current in the
absence of arachidonate and fitted to the Michaelis-Menten equation.
Arachidonate increased the Imax without changing the
glutamate K0.5 value (1.5 µM).
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These results demonstrate that arachidonate increased a
glutamate-dependent rEAAT4 current without affecting
glutamate uptake. This is in contrast to the effects of arachidonate on
the EAAT1 and EAAT2 subtypes, in which L-glutamate uptake
and currents are decreased or increased in parallel (15). Hence, the
ionic nature of the rEAAT4 conductance increased by arachidonate was
investigated further. Similar to the human EAAT1-EAAT4 subtypes (20,
22), rat EAAT4 mediates an uncoupled Cl
conductance in
addition to the sodium-coupled glutamate transport current, because the
outward current was abolished when extracellular chloride was
substituted by gluconate (n = 4; data not shown). To
examine whether arachidonate selectively increased the uncoupled Cl
conductance, the voltage dependence of the current
induced by glutamate was examined in the presence and absence of
arachidonate. The arachidonate-dependent current was
inwardly rectifying and did not reverse at the Cl
equilibrium potential (~
20 mV), indicating that the conductance increased by arachidonate was not Cl
-selective (Fig.
3A). To further rule out an
action of arachidonate on the transporter-mediated anion conductance,
extracellular Cl
was substituted by the more permeant ion
NO3
(4, 20). Similar to results with
the human EAAT4 transporter (4), NO3
was more permeant than Cl
. Replacement of extracellular
Cl
by NO3
increased the
glutamate-induced outward current and shifted the reversal potential to
more negative potentials, from
14.8 ± 1.1 to
81.8 ± 1.1 mV (n = 3; Fig. 3B). Coapplication of 100 µM arachidonate with glutamate slightly inhibited the
outward NO3
current, further
supporting that conclusion that the conductance increased by
arachidonate was not anion-selective (Fig. 3B). The reversal
potential of the glutamate-induced current was shifted approximately
+10 mV by arachidonate (from
14.8 ± 1.1 mV to
4.7 ± 2.4 mV, n = 3), and this shift was not influenced by
changing the Na+ gradient by substitution of 48 mM Na+ with choline (n = 3;
data not shown). Hence, the arachidonate-mediated increase of the
L-glutamate current was selective for ions other than
sodium or chloride.

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Fig. 3.
Arachidonate did not enhance the
NO3 -selective transporter anion conductance.
A, voltage dependence of glutamate transport currents
recorded in Cl -containing Ringer. B, glutamate
transport currents in the same group of cells with
NO3 substituted for Cl .
Note the different scales. , control; ,100 µM
arachidonate. Currents represent means ± S.E., n = 3.
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Glutamate transporters mediate a coupled flux of protons with glutamate
(23). To examine whether a proton-selective current was involved in the
arachidonate potentiation of the L-glutamate current,
currents were measured with varying extracellular pH between 6.5 and
8.5. Altering the extracellular proton concentration markedly
influenced voltage dependence of the arachidonate-dependent current. As the extracellular proton concentration increased, the
potential at which the glutamate current recorded in the presence of
arachidonate crossed the control glutamate current shifted to more
positive potentials (Fig. 4). With
extracellular pH at 7.5, close to the value of the intracellular pH
(24), the arachidonate-dependent current crossed the
control current at 2.3 ± 3.9 mV (n = 11), and
this reversal potential changed 53 mV/pH unit (Fig. 4D).
These results indicate that the major component of the conductance
amplified by arachidonate was proton-selective.

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Fig. 4.
Arachidonate enhances a proton-selective
current. Voltage dependence of currents induced by 30 µM glutamate in the presence and absence of 100 µM arachidonate at pH 6.5 (A), 7.5 (B), and 8.5 (C). D, extracellular pH
shifted the potential at which currents in the presence of arachidonate
crossed over the control transport currents. The l shows
least squares fit with slope of 53.4 mV/pH unit.
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DISCUSSION |
The present results show that arachidonic acid activates a
proton-selective conductance during EAAT4-mediated transport of L-glutamate, extending the recognized types of currents
associated with glutamate transporters. In addition to the current
associated with the
Na+/H+/K+-coupled translocation of
glutamate (15), thermodynamically uncoupled Cl
currents
(20, 22, 25, 26) as well as cation leak currents (27, 28) have been
associated with glutamate transport. Different subtypes exhibit
variability in both their anion (20) and cation conductances (28).
Several other neurotransmitter transporters mediate uncoupled proton
currents (29, 30), but EAAT4 is the first glutamate transporter
reported to exhibit this property.
EAAT4 is predominantly localized to the cerebellum, where it is found
on Purkinje cell bodies and dendrites (1, 2, 3). Transporter-mediated
reuptake of glutamate released at climbing and parallel fiber synapses
onto Purkinje cells plays a role in speeding the decay of postsynaptic
responses (31, 32). The transporters participating in this process are
located both in glial cells surrounding the synapse (33, 34) and in the
postsynaptic dendrites and cell body (4, 35). The pharmacological and electrophysiological properties of the synaptically activated transport
current in Purkinje cells suggest that it is mediated in large part by
EAAT4 (4).
Arachidonic acid is liberated by activation of phospholipase
A2 during neuronal activity (5, 36), and it can modulate activity-dependent changes in synaptic strength in Purkinje
cells (18). Whether exogenously applied or generated by depolarization, arachidonic acid increased the amplitude of glutamate
transporter-mediated currents in rat Purkinje cells (5). The amplitude
of the increase seen in Purkinje cells was similar to that reported
here with the exogenously expressed EAAT4. Together these results
suggest that synaptic activity may lead to activation of a glutamate
transporter-mediated proton influx in neurons that express EAAT4.
Because pH strongly influences the activity of many types of ion
channels (for review see Ref. 37), this property of the transporter
could provide an additional mechanism to modulate postsynaptic
responses. Furthermore this phenomenon could contribute to
intracellular acidification during ischemia as a consequence of the
pathological elevation of glutamate and arachidonate.
We thank Yuqin Yang for oocyte preparation
and T. Otis and J. Wadiche for discussions and comments on the
manuscript.