Howard Hughes Medical Institute, Vollum Institute for Advanced Biomedical Research, Oregon Health Sciences University, Portland, Oregon 97201
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ABSTRACT |
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Recent studies of glutamate transporters in the central nervous system indicate that in addition to their fundamental role in mediating neurotransmitter uptake, these proteins may contribute to the modulation of a variety of cellular processes. Activation of the excitatory amino acid (EAA) carriers generates an electrogenic current attibutable to ion-coupled cotransport. In addition to this transport-associated current, a substrate-gated thermodynamically uncoupled anion flux has been identified that has been proposed to dampen neuronal excitability. Arachidonic acid has been reported to modulate a variety of membrane proteins involved in cellular signaling. Here we discuss recent findings that indicate arachidonic acid stimulates a previously uncharacterized proton-selective conductance in the Purkinje cell-specific subtype, EAAT4. The unique channel-like porperties of the EAATs, their unexpected localization, and physiological evidence propose a modulatory role for the EAATs in neuronal signaling and suggest a broader role for glutamate transporters than simply the clearance of synaptically released glutamate. Thus, the identification of this arachidonate-stimulated proton conductance extends the complexity of mechanisms through which glutamate transporters modulate neuronal excitability.
proton conductance; neuronal excitability; glutamate transporter; ligand-gated chloride conductance; arachidonic acid
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ARTICLE |
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EXCITATORY AMINO ACID transporters in the central
nervous system (CNS) maintain extracellular glutamate concentrations
below excitotoxic levels and contribute to the clearance of glutamate released during neurotransmission. At least five
structurally distinct subtypes of human glutamate transporters,
EAAT1-EAAT5 (1, 2, 11), have been identified and characterized by molecular cloning. Transport of substrates by these carriers is proposed to be thermodynamically coupled to the cotransport of two to
three sodium ions (4, 13, 23), one proton (23), and the
countertransport of a potassium ion (4); thus this process is
electrogenic. However, the amount of charge moved when substrates are
applied to these carriers is greater than would be predicted from the
flux of coupled ions and substrate: this additional current elicited
during substrate application arises from a thermodynamically uncoupled
anion flux (for review, see Ref. 17). The relative proportion of the
current generated by ion-coupled substrate transport or the
ligand-gated chloride conductance varies for each cloned EAAT subtype.
For EAAT1, EAAT2, and EAAT3 the chloride flux is a relatively small
component of the currents, whereas for two of the neuronal
transporters, EAAT4 and EAAT5, the currents elicited by substrates are
almost entirely comprised of a gated anion flux. The association
between these two neuronal EAATs and a ligand-gated chloride
conductance suggests a broader role for transporters in regulating
neuronal excitability and signaling mechanisms. Current data support
the idea that the chloride conductance is intrinsic to the
transporters, but there has been no formal structural or biochemical
evidence to suggest whether single or multiple permeation pathways
exist for anions, substrates and cotransported ions and/or whether
accessory proteins are necessary for the generation of the multiple
conductances associated with these carriers (for review, see Ref. 17).
Alternative models for the transporter-associated conductances are
represented in Fig. 1.
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In addition to the ion channel-like properties attributed to members of this transporter family, the cellular and anatomical localization of the different EAAT subtypes is also consistent with novel, unexpected functions for these carriers. Earlier work using synaptosomal preparations, pathway lesioning experiments, and autoradiographic studies of uptake sites had suggested that excitatory amino acid transporters were predominantly localized on presynaptic neurons and in glia surrounding synapses. This notion that neuronal glutamate transporters are distributed on the terminals of glutamatergic neurons has been challenged by recent studies in human and rat brain, which demonstrate that the neuronal carriers identified in the brain, EAAT3 and EAAT4, are not found on axons or presynaptic terminals, but instead are located on the soma and dendrites of neurons (16, 19). Furthermore, EAAT4 is found predominantly on a GABA-ergic cell type, the cerebellar Purkinje cell, and appears concentrated in dendritic areas that receive major glutamatergic inputs. An additional observation linking glutamate carriers to other components of postsynaptic signaling complexes is the observation that EAAT5, a retinal transporter, contains a PDZ-binding motif at its COOH terminus (2). The same motif has been implicated in receptor and ion channel clustering at the synapse and has been shown to mediate the binding of the cytoplasmic COOH terminus of N-methyl-D-aspartate (NMDA) receptor subunits and of Shaker-type potassium channels to a group of abundant synaptic proteins that include PSD95 and PSD93. The apparent postsynaptic localization of the neuronal EAATs and their expression in nonglutamatergic cells suggests that they do not simply serve as sites of neurotransmitter reuptake and may function in other ways.
Arachidonic acid released during neuronal activity alters the
electrical and biochemical properties of a variety of membrane proteins
involved in cellular signaling including several
neurotransmitter transporters. For the glutamate transporters EAAT1,
EAAT2, and EAAT3, arachidonic acid was reported to modulate the
kinetics of substrate transport as assessed by uptake of radiolabeled
glutamate and/or by measurement of substrate-elicited currents (22).
Arachidonic acid appears to have very different actions on the function
of EAAT4. In EAAT4-expressing oocytes, the application of
physiologically relevant concentrations of arachidonic acid produces an
approximately twofold stimulation in the substrate-gated currents at
60 mV which cannot be attributed to the modulation of substrate
transport or of the ligand-gated anion current associated with this
carrier (10). In addition, preliminary studies in our
laboratory suggest that a similar stimulation of the substrate-gated
current is observed when arachidonic acid is applied to the retinal
carrier, EAAT5. In the case of EAAT4, arachidonic acid stimulates a
novel ligand-activated conductance selective for protons. This effect
does not require the metabolism of arachidonic acid, and is not blocked
by inhibitors of endogenous oocyte ion exchangers (10). This
observation that arachidonic acid stimulates a proton conductance
associated with EAAT4 expands the repertoire of ion channel-like
properties associated with the EAAT family, and suggests yet another
mechanism, perhaps involving local pH changes, through which synaptic
excitability can be modulated by substrate binding and transport.
Actions of arachidonic acid on EAAT4.
The effect of arachidonic acid on EAAT4-expressing oocytes was assessed
by measuring substrate-gated currents and radiolabeled uptake. At
60 mV, arachidonic acid stimulated
L-Asp-gated currents 200 ± 44% (n = 56) (Fig. 2A),
with an EC50 of 1.7 ± 0.3 µM. Several lines of evidence suggest that this stimulation is a
direct consequence of the application of arachidonic acid and not the
result of arachidonic acid metabolism. Arachidonic acid was able to
potently stimulate the
L-Asp-gated current in the
presence of inhibitors of lipoxygenase, cyclooxygenase, and
monooxygenase inhibitors. Other polyunsaturated fatty
acids were able to stimulate the
L-Asp-gated current with a rank
order of potency that paralleled their degree of unsaturation
(docosahexaenoic acid > arachidonic acid > linoleic acid > linolenic acid > oleic acid). In addition, the inactive analog of
arachidonic acid, arachidonic acid ethyl ester, was inactive in cells
that showed a subsequent stimulation by arachidonic acid.
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Previous work demonstrated that with EAAT1, arachidonic acid decreased the maximal uptake velocity, and with EAAT2, it increased the affinity for L-glutamate with no change in the maximal transport rate (22). Surprisingly, although arachidonic acid potentiated substrate-activated currents in EAAT4-expressing oocytes, it had little effect on transport activity. No significant differences in either the affinity or the maximal transport rate for L-[3H]Asp was observed in the presence or absence of a maximal concentration of arachidonic acid (100 µM). Similarly, arachidonic acid had no effects on the kinetics or sodium dependence of the currents induced by L-glutamate (10).
Additional experiments ruled out the possibility that the stimulation
of the L-Asp-induced current by
arachidonic acid might be caused by a stimulation of the ligand-gated
anion conductance. As shown in the current-voltage relation in Fig.
2B, the coapplication of arachidonic
acid produced robust increases in the current amplitude at the most
negative potentials, with no apparent stimulation of outward currents
at potentials more positive than the reversal potential
(Erev).
In cells in which the chloride equilibrium potential, ECl, was directly measured to be
14 ± 0.4 mV, the coapplication of arachidonic acid shifted
Erev of the
L-Asp-induced current to
4.2 ± 1 mV (n = 5), away
from ECl.
Furthermore, equimolar substitution of extracellular chloride with the
impermeant ion gluconate completely abolished the outward current
induced by L-Asp, whereas
coapplication of arachidonic acid and
L-Asp still elicited an outward
current (Fig. 2C). Varying
extracellular chloride concentration (extracellular
[Cl
]) from 104 to 14 mM produced a shift in
Erev of
57 ± 2 mV per 10-fold change in
[Cl
]
(n = 7), whereas varying extracellular
[Cl
] in the
presence of arachidonic acid produced only a modest shift in
Erev. Finally,
arachidonic acid was still able to shift
Erev to more
positive potentials and stimulate the amplitude of the L-Asp-induced current at
potentials more negative than
Erev when extracellular chloride was replaced with nitrate (10), a more permeant
anion (20). These results provide compelling evidence that the
arachidonic acid-stimulated conductance is not mediated by chloride ions.
Arachidonic acid stimulates a novel proton-selective
conductance. Ion substitution experiments indicate that
arachidonic acid stimulates a conductance that is selective for
protons. Varying extracellular sodium, calcium, and potassium ions had
no significant effects on the
Erev of the
current stimulated by the coapplication of arachidonic acid and
L-Asp. Varying extracellular
proton concentration from pH 6.5 to 8.5 had no effect on the
Erev of the
L-Asp-induced current, but
significantly altered the amplitude of the current. In contrast, the
coapplication of arachidonic acid produced a marked shift in
Erev with
alterations in extracellular pH, indicating the protons (or
OH) is likely to be the
charge-carrying species of the arachidonic acid-induced current. To
more clearly establish the proton selectivity of the arachidonic
acid-stimulated current, substrate-elicited currents in
EAAT4-expressing oocytes were studied under chloride-depleted conditions. The coapplication of arachidonic acid and
L-Asp in gluconate-substituted
Ringer produced a shift of
38 ± 0.3 mV per pH unit when
extracellular pH was varied from 6.5 to 8.5 (Fig. 2,
D-F)
(10). Unexpectedly, under these conditions, similar pH-dependent shifts
for the L-Asp-induced currents
roughly paralleled those observed for the coapplication of arachidonic
acid with substrate, suggesting that
L-Asp may gate a proton flux
that is stimulated by exogenous arachidonic acid.
Deuterium substitution has been used to investigate proton flux through
a variety of channels (6, 7). Because deuterons exhibit reduced
permeability relative to protons, whereas the mobility of
O2H
and OH
is
comparable, we examined the effect of
2H2O
substitution on both the L-Asp-
and arachidonic acid-stimulated current. No significant effects on
[3H]L-Asp
flux were observed in the presence of
2H2O-substituted
Ringer solution. In contrast, although 80%
2H2O
substitution produced no significant alterations in the
L-Asp-induced current, the
arachidonic acid-stimulated current was completely abolished (Fig.
2G) in cells showing a significant
stimulation of the L-Asp-induced
current with the subsequent application of arachidonic acid in
proton-rich Ringer (10).
To rule out the possibility that endogenous oocyte ion exchangers
participate in the arachidonic acid-stimulated proton conductance, a
variety of inhibitors of endogenous oocyte ion exchangers were tested
for their ability to inhibit the stimulation of the
L-Asp-induced current by
arachidonic acid. No significant diminution in the stimulation of the
L-Asp-induced current was
observed in the presence of inhibitors of the
Na+/H+
exchanger, the
Na+/K+/2Cl
exchanger or the
Na+-K+-ATPase
(10), suggesting that the activation of the proton conductance is
likely to be mediated directly through EAAT4.
Physiological significance of EAAT-associated ion
conductances. Emerging data indicate that transporters
and their associated conductances may contribute to intercellular
signaling in the nervous system beyond the canonical role of the
carriers in terminating synaptic chemical signals through transmitter
reuptake and recycling. In retinal bipolar cells, a transporter-gated
chloride conductance has been proposed to mediate the cone component of
the ON bipolar cell light response (12). Cone
photoreceptor cells from the tiger salamander respond to the glutamate
they release with hyperpolarizing responses through the activation of a
glutamate-gated Cl
conductance; this current appears to act as feedback mechanism to limit
further depolarization and consequent glutamate release (15). Evidence
for a retinal glutamate transporter with an associated glutamate-gated
Cl
conductance has come
from molecular cloning studies in retina, which have identified and
characterized at least seven distinct glutamate transporter subtypes
from salamander retina (9). A human homolog of one of the
retinal-specific carriers, human EAAT5, also appears to have a
substantial substrate-activated chloride current and is a promising
candidate for the carrier that serves as a glutamate sensor in
photoreceptors. In other brain regions, such as the Purkinje cells that
express EAAT4, the activation of a
Cl
conductance has been
proposed to dampen neuronal excitability. As discussed above,
arachidonic acid further modulates the function of EAAT4 to stimulate
an additional substrate-gated conductance carried predominantly by
protons (10).
EAAT4 is concentrated postsynaptically in the extrajunctional spaces of
climbing fiber- and parallel fiber-Purkinje cell synapses (19). In a
recent patch-clamp study using rat cerebellar Purkinje cells, Kataoka
et al. (14) found that either sustained depolarization or exogenous
application of arachidonic acid induced an approximately twofold increase in the maximum current mediated by the EAATs. This
increase was blocked by inhibitors of phospholipase
A2 or phospholipase C but not
protein kinase C or
Ca2+/calmodulin-dependent kinase
(Fig 3). Sustained depolarization was
proposed to activate arachidonic acid release and stimulate the
EAAT4-associated anion conductance by increasing the affinity for
L-Asp. Although
L-Asp clearly activated an anion
conductance in these cells, the identity of the charge-carrying species
underlying the increased current observed with arachidonic acid was not
formally demonstrated. Since our studies imply that arachidonic acid
stimulates a proton conductance associated with the Purkinje cell
transporter EAAT4, the transporter-mediated currents observed in
Purkinje cell cultures with sustained depolarization or the exogenous
application of arachidonic acid are likely to be mediated by protons.
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The multiple functional properties associated with the EAAT4 carrier are particularly interesting when considering the selective postsynaptic localization of EAAT4 in cerebellar Purkinje neurons. Inhibition of EAAT transporters at climbing fiber and parallel fiber-Purkinje cell synapses has been reported to slow the decay time and decrease the amplitude of the EPSC (18). At these synapses the coactivation of AMPA and metabotropic glutamate receptors and the concurrent increase in cytosolic calcium concentration has been proposed to be an important mechanism for arachidonic acid release (8). In addition, increased levels of arachidonic acid have been implicated in the induction of long-term depression (LTD) in this region and have been reported to increase in pathological conditions (3). Alterations in pH have been proposed to be an important means of regulating many cellular functions. Although it is difficult to predict whether the activation of a proton conductance through EAAT4 might acidify the intracellular milieu of a Purkinje cell dendrite sufficiently to modulate the activity of pH-sensitive cellular functions, the complex geometry of the dendrite makes it conceivable that local alterations in intracellular pH might modulate other proteins involved in synaptic excitability, such as glutamate and GABA receptors (5). Thus the complex properties associated with EAAT4 provide several mechanisms by which activation of this protein might modulate neuronal excitability: the termination of synaptic transmission by glutamate transport, the dampening of cellular excitability with its ligand-gated anion conductance, and the stimulation of a proton conductance by arachidonic acid could all serve as mechanisms by which to regulate neuronal excitability.
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FOOTNOTES |
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This article is the first of five in this forum, which is based on a series of reports on glutamate transport and glutamate metabolism that was first presented at Experimental Biology '98 in San Francisco, CA.
Address for reprint requests and other correspondence: S. G. Amara, HHMI, Vollum Institute, Oregon Health Sciences University, L474, 3181 Sam Jackson Park Rd., Portland, Oregon 97201-3098 (E-mail: amaras{at}ohsu.edu).
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REFERENCES |
---|
![]() ![]() ![]() ![]() |
---|
1.
Arriza, J. L.,
W. A. Fairman,
J. I. Wadiche,
G. H. Murdoch,
M. P. Kavanaugh,
and
S. G. Amara.
Functional comparisons of three glutamate transporter subtypes cloned from human motor cortex.
J. Neurosci.
14:
5559-5569,
1994[Abstract].
2.
Arriza, J. L.,
S. Eliasof,
M. P. Kavanaugh,
and
S. G. Amara.
Excitatory amino acid transporter 5, a retinal glutamate transporter coupled to a chloride conductance.
Proc. Natl. Acad. Sci. USA
94:
4155-4160,
1997
3.
Attwell, D.,
B. Miller,
and
M. Sarantis.
Arachidonic acid as a messenger in the central nervous system.
Neuroscience
5:
159-169,
1993.
4.
Barbour, B.,
H. Brew,
and
D. Attwell.
Electrogenic glutamate uptake in glial cells is activated by intracellular potassium.
Nature
335:
433-435,
1988[Medline].
5.
Chesler, M.,
and
K. Kaila.
Modulation of pH by neuronal activity.
Trends Neurosci.
15:
396-402,
1992[Medline].
6.
Deamer, D. W.
Proton permeation of lipid bilayers.
J. Bioenerg. Biomembr.
19:
457-479,
1987[Medline].
7.
Decoursey, T. E.,
and
V. V. Cherny.
Deuterium isotope effects on permeation and gating of proton channels in rat alveolar epithelium.
J. Gen. Physiol.
109:
415-434,
1997
8.
Dumuis, A.,
J. P. Pin,
K. Oomagari,
M. Sebben,
and
J. Bockaert.
Arachidonic acid released from striatal neurons by joint stimulation of ionotropic and metabotropic quisqualate receptors.
Nature
347:
182-184,
1990[Medline].
9.
Eliasof, S.,
J. L. Arriza,
B. H. Leighton,
M. P. Kavanaugh,
and
S. G. Amara.
Excitatory amino acid transporters of the salamander retina: identification, localization, and function.
J. Neurosci.
18:
698-712,
1998
10.
Fairman, W. A.,
M. S. Sonders,
G. H. Murdoch,
and
S. G. Amara.
Arachidonic acid elicits a substrate-gated proton current associated with the glutamate transporter EAAT4.
Nature Neuroscience
1:
105-113,
1998.[Medline]
11.
Fairman, W. A.,
R. J. Vandenberg,
J. L. Arriza,
M. P. Kavanaugh,
and
S. G. Amara.
An excitatory amino-acid transporter with properties of a ligand-gated chloride channel.
Nature
375:
599-603,
1995[Medline].
12.
Grant, G. B.,
and
J. E. Dowling.
A glutamate-activated chloride current in cone-driven ON bipolar cells of the white perch retina.
J. Neurosci.
15:
3852-3862,
1995[Abstract].
13.
Kanai, Y.,
S. Nussberger,
M. F. Romero,
W. F. Boron,
S. C. Hebert,
and
M. A. Hediger.
Electrogenic properties of the epithelial and neuronal high affinity glutamate transporter.
J. Biol. Chem.
270:
16561-16568,
1995
14.
Kataoka, Y.,
H. Morii,
Y. Watanabe,
and
H. Ohmori.
A postsynaptic excitatory amino acid transporter with chloride conductance functionally regulated by neuronal activity in cerebellar Purkinje cells.
J. Neurosci.
17:
7017-7024,
1997
15.
Picaud, S.,
H. P. Larsson,
D. P. Wellis,
H. Lecar,
and
F. Werblin.
Cone photoreceptors respond to their own glutamate release in the tiger salamander.
Proc. Natl. Acad. Sci. USA
92:
9417-9421,
1995[Abstract].
16.
Rothstein, J. D.,
L. Martin,
A. I. Levey,
M. Dykes-Hoberg,
L. Jin,
D. Wu,
N. Nash,
and
R. W. Kuncl.
Localization of neuronal and glial glutamate transporters.
Neuron
13:
713-725,
1994[Medline].
17.
Sonders, M. S.,
and
S. G. Amara.
Channels in transporters.
Curr. Opin. Neurobiol.
6:
294-302,
1996[Medline].
18.
Takahashi, M.,
Y. Kovalchuk,
and
D. Attwell.
Pre- and postsynaptic determinants of EPSC waveform at cerebellar climbing fiber and parallel fiber to Purkinje cell synapses.
J. Neurosci.
15:
5693-5702,
1995[Abstract].
19.
Tanaka, J.,
R. Ichikawa,
M. Watanabe,
K. Tanaka,
and
Y. Inoue.
Extra-junctional localization of glutamate transporter EAAT4 at excitatory Purkinje cell synapses.
Neuroreport
8:
2461-2464,
1997[Medline].
20.
Wadiche, J. I.,
S. G. Amara,
and
M. P. Kavanaugh.
Ion fluxes associated with excitatory amino acid transport.
Neuron
15:
721-728,
1995[Medline].
21.
Yamada, K.,
M. Watanabe,
T. Shibata,
K. Tanaka,
K. Wada,
and
Y. Inoue.
EAAT4 is a postsynaptic glutamate transporter at Purkinje cell synapses.
Neuroreport
7:
2013-2017,
1996[Medline].
22.
Zerangue, N.,
J. L. Arriza,
S. G. Amara,
and
M. P. Kavanaugh.
Differential modulation of human glutamate transporter subtypes by arachidonic acid.
J. Biol. Chem.
270:
6433-6435,
1995
23.
Zerangue, N.,
and
M. P. Kavanaugh.
Flux coupling in a neuronal glutamate transporter.
Nature
383:
634-637,
1996[Medline].