Neuronal Glutamate Uptake Research Articles

Allosteric gate modulation confers K+ coupling in glutamate transporters

Excitatory amino acid transporters (EAATs) mediate glial and neuronal glutamate uptake to terminate synaptic transmission and to ensure low resting glutamate concentrations. Effective glutamate uptake is achieved by cotransport with 3 Na+ and 1 H+, in exchange with 1 K+. The underlying principles of this complex transport stoichiometry remain poorly understood. We use molecular dynamics simulations and electrophysiological experiments to elucidate how mammalian EAATs harness K+ gradients, unlike their K+‐independent prokaryotic homologues. Glutamate transport is achieved via elevator‐like translocation of the transport domain. In EAATs, glutamate‐free re‐translocation is prevented by an external gate remaining open until K+ binding closes and locks the gate. Prokaryotic GltPh contains the same K+‐binding site, but the gate can close without K+. Our study provides a comprehensive description of K+‐dependent glutamate transport and reveals a hitherto unknown allosteric coupling mechanism that permits adaptions of the transport stoichiometry without affecting ion or substrate binding.

Dysregulation of synaptic and extrasynaptic N-methyl-D-aspartate receptors induced by amyloid-β

The toxicity of amyloid-beta (Aβ) is strongly associated with Alzheimer's disease (AD), which has a high incidence in the elderly worldwide. Recent evidence showed that alteration in the activity of N-methyl-D-aspartate receptors (NMDARs) plays a key role in Aβ-induced neurotoxicity. However, the activation of synaptic and extrasynaptic NMDARs has distinct consequences for plasticity, gene regulation, neuronal death, and Aβ production. This review focuses on the dysregulation of synaptic and extrasynaptic NMDARs induced by Aβ. On one hand, Aβ downregulates the synaptic NMDAR response by promoting NMDAR endocytosis, leading to either neurotoxicity or neuroprotection. On the other hand, Aβ enhances the activation of extrasynaptic NMDARs by decreasing neuronal glutamate uptake and inducing glutamate spillover, subsequently causing neurotoxicity. In addition, selective enhancement of synaptic activity by low doses of NMDA, or reduction of extrasynaptic activity by memantine, a non-competitive NMDAR antagonist, halts Aβ-induced neurotoxicity. Therefore, future neuroprotective drugs for AD should aim at both the enhancement of synaptic activity and the disruption of extrasynaptic NMDAR-dependent death signaling.

Impairment of Neuronal Glutamate Uptake and Modulation of the Glutamate Transporter GLT-1 Induced by Retinal Ischemia

Excitotoxicity has been implicated in the retinal neuronal loss in several ocular pathologies including glaucoma. Dysfunction of Excitatory Amino Acid Transporters is often a key component of the cascade leading to excitotoxic cell death. In the retina, glutamate transport is mainly operated by the glial glutamate transporter GLAST and the neuronal transporter GLT-1. In this study we evaluated the expression of GLAST and GLT-1 in a rat model of acute glaucoma based on the transient increase of intraocular pressure (IOP) and characterized by high glutamate levels during the reperfusion that follows the ischemic event associated with raised IOP. No changes were reported in GLAST expression while, at neuronal level, a reduction of glutamate uptake and of transporter reversal-mediated glutamate release was observed in isolated retinal synaptosomes. This was accompanied by modulation of GLT-1 expression leading to the reduction of the canonical 65 kDa form and upregulation of a GLT-1-related 38 kDa protein. These results support a role for neuronal transporters in glutamate accumulation observed in the retina following an ischemic event and suggest the presence of a GLT-1 neuronal new alternative splice variant, induced in response to the detrimental stimulus.

The Discovery of Slowness: Low-Capacity Transport and Slow Anion Channel Gating by the Glutamate Transporter EAAT5

Excitatory amino acid transporters (EAATs) control the glutamate concentration in the synaptic cleft by glial and neuronal glutamate uptake. Uphill glutamate transport is achieved by the co-/countertransport of Na+ and other ions down their concentration gradients. Glutamate transporters also display an anion conductance that is activated by the binding of Na+ and glutamate but is not thermodynamically coupled to the transport process. Of the five known glutamate transporter subtypes, the retina-specific subtype EAAT5 has the largest conductance relative to glutamate uptake activity. Our results suggest that EAAT5 behaves as a slow-gated anion channel with little glutamate transport activity. At steady state, EAAT5 was activated by glutamate, with a Km= 61 ± 11 μM. Binding of Na+ to the empty transporter is associated with a Km = 229 ± 37 mM, and binding to the glutamate-bound form is associated with a Km = 76 ± 40 mM. Using laser-pulse photolysis of caged glutamate, we determined the pre-steady-state kinetics of the glutamate-induced anion current of EAAT5. This was characterized by two exponential components with time constants of 30 ± 1 ms and 200 ± 15 ms, which is an order of magnitude slower than those observed in other glutamate transporters. A voltage-jump analysis of the anion currents indicates that the slow activation behavior is caused by two slow, rate-limiting steps in the transport cycle, Na+ binding to the empty transporter, and translocation of the fully loaded transporter. We propose a kinetic transport scheme that includes these two slow steps and can account for the experimentally observed data. Overall, our results suggest that EAAT5 may not act as a classical high-capacity glutamate transporter in the retina; rather, it may function as a slow-gated glutamate receptor and/or glutamate buffering system.

Region- and pattern-specific effects of glutamate uptake blockers on epileptiform activity in rat brain slices

Many epileptic syndromes develop into pharmaco-resistant forms, calling for the development of new anticonvulsant strategies. The transmitter glutamate serves a double role as excitatory transmitter and as precursor for GABA, thus interfering with glutamate uptake may therefore exert complex effects on excitation-inhibition-balance in epileptic networks. In the present study we tested the effect of two different glutamate uptake blockers on acutely induced epileptiform activity in hippocampal-entorhinal cortex slices from adult rats: dihydrokainate (DHK) which blocks predominantly glial glutamate uptake, and threo-beta-benzyloxyaspartic acid (TBOA) which blocks both glial and neuronal glutamate uptake. Three different models were used to induce epileptiform discharges: (i) increasing NMDA receptor-mediated excitation by omitting Mg(2+)-ions; (ii) blocking potassium channels by 4-aminopyridine; (iii) reducing GABA(A) receptor-mediated inhibition by penicillin. Application of DHK or TBOA markedly reduced the frequency of epileptiform discharges in CA1 in the low magnesium and the 4-AP model while pathological activity was increased in the penicillin-model. In contrast, frequency of epileptiform discharges in EC was consistently increased by DHK and TBOA. Effects of DHK were more easily reversible than those of TBOA. Thus glutamate uptake blockers exert variable effects on epileptiform activity, depending on brain region and on the mechanism of ictogenesis.

Soluble Oligomers of Amyloid β Protein Facilitate Hippocampal Long-Term Depression by Disrupting Neuronal Glutamate Uptake

In Alzheimer's disease (AD), the impairment of declarative memory coincides with the accumulation of extracellular amyloid-beta protein (Abeta) and intraneuronal tau aggregates. Dementia severity correlates with decreased synapse density in hippocampus and cortex. Although numerous studies show that soluble Abeta oligomers inhibit hippocampal long-term potentiation, their role in long-term synaptic depression (LTD) remains unclear. Here, we report that soluble Abeta oligomers from several sources (synthetic, cell culture, human brain extracts) facilitated electrically evoked LTD in the CA1 region. Abeta-enhanced LTD was mediated by mGluR or NMDAR activity. Both forms of LTD were prevented by an extracellular glutamate scavenger system. Abeta-facilitated LTD was mimicked by the glutamate reuptake inhibitor TBOA, including a shared dependence on extracellular calcium levels and activation of PP2B and GSK-3 signaling. In accord, synaptic glutamate uptake was significantly decreased by soluble Abeta. We conclude that soluble Abeta oligomers perturb synaptic plasticity by altering glutamate recycling at the synapse and promoting synapse depression.

Glycinergic Transmission Shaped by the Corelease of GABA in a Mammalian Auditory Synapse

The firing pattern of neurons is shaped by the convergence of excitation and inhibition, each with finely tuned magnitude and duration. In an auditory brainstem nucleus, glycinergic inhibition features fast decay kinetics, the mechanism of which is unknown. By applying glycine to native or recombinant glycine receptors, we show that response decay times are accelerated by addition of GABA, a weak partial agonist of glycine receptors. Systematic variation in agonist exposure time revealed that fast synaptic time course may be achieved with submillisecond exposures to mixtures of glycine and GABA at physiological concentrations. Accordingly, presynaptic terminals generally contained both transmitters, and depleting terminals of GABA slowed glycinergic synaptic currents. Thus, coreleased GABA accelerates glycinergic transmission by acting directly on glycine receptors, narrowing the time window for effective inhibition. Packaging both weak and strong agonists in vesicles may be a general means by which presynaptic neurons regulate the duration of postsynaptic responses.

Effects of glutamate transporter and receptor ligands on neuronal glutamate uptake

The excitatory amino acids (EAAs) transporters regulate the balance between physiological and pathological signaling over stimulation of the glutamatergic system pathway. The effect of transportable substrates and glutamate (Glu) receptor agonists on Glu uptake in neuronal cells was assessed at different conditions. Cells pre-incubated with Glu, l- or d-aspartate (Asp) and washed presented an inhibition on [ 3H]-Glu uptake and this effect was not mimicked by Glu receptors agonists. The effects of l- and d-Asp were not altered by the presence of N-methyl- d-aspartate (NMDA) receptor antagonists. Thus, the reduction on Glu uptake induced by EAAs is probably linked to the transporter activity. In contrast, the presence of NMDA or (1 S,3 R)-1-aminocyclopentane-1,3-dicarboxylic acid (SR-ACPD) during the pre-incubation and the [ 3H]-Glu uptake assay period increased Glu uptake, whilst kainic acid (KA) had no effect. The NMDA effect was not altered by its antagonists (±)-2-amino-5-phosphonopentanoic acid (AP-5) or dizocilpine (MK-801). The SR-ACPD effect was due to the activation of metabotropic Glu receptor, since it was abolished by its antagonist, l(±)-2-amino-3-phosphonopropionic acid ( l-AP3). Thus, the current studies suggest that the neuronal EAAs transporter is regulated in different manner by transportable substrates and Glu receptor agonists. The possible involvement of this modulation after certain neurotoxicity insults is discussed.

Role of glutamine and neuronal glutamate uptake in glutamate homeostasis and synthesis during vesicular release in cultured glutamatergic neurons

Glutamate exists in a vesicular as well as a cytoplasmic pool and is metabolically closely related to the tricarboxylic acid (TCA) cycle. Glutamate released during neuronal activity is most likely to a large extent accumulated by astrocytes surrounding the synapse. A compensatory flux from astrocytes to neurons of suitable precursors is obligatory as neurons are incapable of performing a net synthesis of glutamate from glucose. Glutamine appears to play a major role in this context. Employing cultured cerebellar granule cells, as a model system for glutamatergic neurons, details of the biosynthetic machinery have been investigated during depolarizing conditions inducing vesicular release. [U- 13C]Glucose and [U- 13C]glutamine were used as labeled precursors for monitoring metabolic pathways by nuclear magnetic resonance (NMR) spectroscopy and liquid chromatography–mass spectrometry (LC-MS) technologies. To characterize release mechanisms and influence of glutamate transporters on maintenance of homeostasis in the glutamatergic synapse, a quantification was performed by HPLC analysis of the amounts of glutamate and aspartate released in response to depolarization by potassium (55 mM) in the absence and presence of dl-threo-β-benzyloxyaspartate (TBOA) and in response to l-trans-pyrrolidine-2,4-dicarboxylate (t-2,4-PDC), a substrate for the glutamate transporter. Based on labeling patterns of glutamate the biosynthesis of the intracellular pool of glutamate from glutamine was found to involve the TCA cycle to a considerable extent (approximately 50%). Due to the mitochondrial localization of PAG this is unlikely only to reflect amino acid exchange via the cytosolic aspartate aminotransferase reaction. The involvement of the TCA cycle was significantly lower in the synthesis of the released vesicular pool of glutamate. However, in the presence of TBOA, inhibiting glutamate uptake, the difference between the intracellular and the vesicular pool with regard to the extent of involvement of the TCA cycle in glutamate synthesis from glutamine was eliminated. Surprisingly, the intracellular pool of glutamate was decreased after repetitive release from the vesicular pool in the presence of TBOA indicating that neuronal reuptake of released glutamate is involved in the maintenance of the neurotransmitter pool and that 0.5 mM glutamine exogenously supplied is inadequate to sustain this pool.

Presenilin-1 and intracellular calcium stores regulate neuronal glutamate uptake.

Glutamate uptake by high affinity glutamate transporters is essential for preventing excitotoxicity and maintaining normal synaptic function. We have discovered a novel role for presenilin-1 (PS1) as a regulator of glutamate transport. PS1-deficient neurons showed a decrease in glutamate uptake of approximately 50% compared to wild-type neurons. Gamma-secretase inhibitor treatment mimicked the effects of PS1 deficiency on glutamate uptake. PS1 loss-of-function, accomplished by PS1 deficiency or gamma-secretase inhibitor treatment, caused a corresponding decrease in cell surface expression of the neuronal glutamate transporter, EAAC1. PS1 deficiency is known to reduce intracellular calcium stores. To explore the possibility that PS1 influences glutamate uptake via regulation of intracellular calcium stores, we examined the effects of treating neurons with caffeine, thapsigargin, and SKF-96365. These compounds depleted intracellular calcium stores by distinct means. Nonetheless, each treatment mimicked PS1 loss-of-function by impairing glutamate uptake and reducing EAAC1 expression at the cell surface. Blockade of voltage-gated calcium channels, activation and inhibition of protein kinase C (PKC), and protein kinase A (PKA) all had no effect on glutamate uptake in neurons. Taken together, these findings indicate that PS1 and intracellular calcium stores may play a significant role in regulating glutamate uptake and therefore may be important in limiting glutamate toxicity in the brain.

Hypoxia stimulates glutamate uptake in whole rat retinal cells in vitro

Glutamate uptake in neurons and glial cells is essential to prevent the persistence of excitotoxic levels of glutamate observed during ischemia. We demonstrated that a short period of hypoxia stimulated the apparent glutamate transport rate in isolated rat retinal cells. The observed increase in glutamate uptake was not affected by glutamate receptor antagonists, protein kinase inhibitors, antioxidant or neo-synthesis inhibitors. However, inhibition of actin polymerization reversed the hypoxia-induced increase in glutamate uptake, suggesting a mobilization of transporters to the cell membrane. Moreover, the depletion in cell glutathione stimulated in the same manner the glutamate uptake and emphasized the key role of glutamate in the control of the level of this antioxidant. This rapid up-regulation of glutamate transport could be considered as an adaptative mechanism of neuroprotection.

Neuronal Glutamate Uptake Contributes to GABA Synthesis and Inhibitory Synaptic Strength

Neurons must maintain a supply of neurotransmitter in their presynaptic terminals to fill synaptic vesicles. GABA is taken up into inhibitory terminals by transporters or is synthesized from glutamate by glutamic acid decarboxylase. Here we report that glutamate transporters supply GABAergic terminals in the hippocampus with glutamate, which is then used to synthesize GABA for filling synaptic vesicles. Glutamate transporter antagonists reduced IPSC and miniature IPSC (mIPSC) amplitudes, consistent with a reduction in the amount of GABA packaged into each synaptic vesicle. This reduction occurred rapidly and independently of synaptic activity, suggesting that modulation of vesicular GABA content does not require vesicle release and refilling. Raising extracellular glutamate levels increased mIPSC amplitudes by enhancing glutamate uptake and, consequently, GABA synthesis. These results indicate that neuronal glutamate transporters strengthen inhibitory synapses in response to extracellular glutamate. This modulation appears to occur under normal conditions and may constitute a negative feedback mechanism to combat hyperexcitability.

Long-term potentiation and contextual fear conditioning increase neuronal glutamate uptake.

Induction and expression of long-term potentiation (LTP) in area CA1 of the hippocampus require the coordinated regulation of several cellular processes. We found that LTP in area CA1 was associated with an N-methyl-D-aspartate (NMDA) receptor-dependent increase in glutamate uptake. The increase in glutamate uptake was inhibited by either removal of Na+ or addition of D,L-threo-beta-hydroxyaspartate. Dihydrokainate (DHK), a specific inhibitor of the glial glutamate transporter GLT-1, did not block the increase in glutamate uptake. LTP was also associated with a translocation of the EAAC1 glutamate transporter from the cytosol to the plasma membrane. Contextual fear conditioning increased the maximum rate (Vmax) of glutamate uptake and membrane expression of EAAC1 in area CA1. These results indicate that regulation of glutamate uptake may be important for maintaining the level of synaptic strength during long-term changes in synaptic efficacy.

Effects of thiopental on transport and metabolism of glutamate in cultured cerebellar granule neurons

This study was performed to analyze the effects of the barbiturate thiopental on neuronal glutamate uptake, release and metabolism. Since barbiturates are known to bind to the GABA A receptor, some experiments were carried out in the presence of GABA. Cerebellar granule neurons were incubated for 2 h in medium containing 0.25 mM [U- 13C]glutamate, 3 mM glucose, 50 μM GABA and 0.1 or 1 mM thiopental when indicated. When analyzing cell extracts, it was surprisingly found that in addition to glutamate, aspartate and glutathione, GABA was also labeled. In the medium, label was observed in glutamate, aspartate and lactate. Glutamate exhibited different labeling patterns, indicating metabolism in the tricarboxylic acid cycle, and subsequent release. A net uptake of [U- 13C]glutamate and unlabeled glucose was seen under all conditions. The amounts of most metabolites synthesized from [U- 13C]glutamate were unchanged in the presence of GABA with or without 0.1 mM thiopental. In the presence of 1 mM thiopental, regardless of the presence of GABA, decreased amounts of [1,2,3- 13C]glutamate and [U- 13C]aspartate were found in the medium. In the cell extracts increased [U- 13C]glutamate, [1,2,3- 13C]glutamate, labeled glutathione and [U- 13C]aspartate were observed in the 1 mM thiopental groups. Glutamate efflux and uptake were studied using [ 3H] d-aspartate. While efflux was substantially reduced in the presence of 1 mM thiopental, this barbiturate only marginally inhibited uptake even at 3 mM. These results may suggest that the previously demonstrated neuroprotective action of thiopental could be related to its ability to reduce excessive glutamate outflow. Additionally, thiopental decreased the oxidative metabolism of [U- 13C]glutamate but at the same time increased the detectable metabolites derived from the TCA cycle. These latter effects were also exerted by GABA.

Non-synaptic localization of the glutamate transporter EAAC1 in cultured hippocampal neurons.

It has been postulated for several years that the high affinity neuronal glutamate uptake system plays a role in clearing glutamate from the synaptic cleft. Four different glutamate transporter subtypes are now identified, the major neuronal one being EAAC1. To be a good candidate for the reuptake of glutamate at the synaptic cleft, EAAC1 should be properly located at synapses, either at pre- or postsynaptic sites. We have investigated the distribution of EAAC1 in primary cultures of hippocampal neurons, which represent an advantageous model for the study of synaptogenesis and synaptic specializations. We have demonstrated that EAAC1 immunoreactivity is segregated in the somatodendritic compartment of fully differentiated hippocampal neurons, where it is localized in the dendritic shaft and in the spine neck, outside the area facing the active zone. No co-localization of EAAC1 immunoreactivity with the stainings produced by typical presynaptic and postsynaptic markers was ever observed, indicating that EAAC1 is not to be considered a synaptic protein. Accordingly, the developmental pattern of expression of EAAC1 was found to be different from that of typical synaptic markers. Moreover, EAAC1 was expressed in the somatodendritic compartment of hippocampal neurons already at stages preceding the formation of synaptic contacts, and was also expressed in GABAergic interneurons with identical subcellular distribution. Taken together, these data rule against a possible role for EAAC1 in the clearance of glutamate from within the cleft and in the regulation of its time in the synapse. They suggest an unconventional non-synaptic function of this high-affinity glutamate carrier, not restricted to glutamatergic fibres.