Excitatory amino acid transporters as potential drug targets

Background: Excitatory amino acid transporters (EAATs) are transmembrane proteins responsible for the uptake of (S)-glutamate (Glu) from the synaptic cleft, thereby terminating the glutamatergic neurotransmitter signal. Today five subtypes have been identified. Except for EAAT2, their individual roles or functions in the CNS are yet to be fully understood due to the shortage of subtype-selective ligands. Objective/methods: We examine the latest developments in this field by addressing EAAT expression pattern, localization and pharmacology. We present highlights of published work on inhibitors as well as enhancers which display subtype preference or selectivity and discuss which pathological conditions in the CNS such ligands may be beneficial to. Results/conclusions: Not until subtype-selective enhancers, inhibitors and substrates for all five EAAT subtypes have been discovered can a full and detailed understanding of EAATs be obtained. Thus we encourage collaboration between organic chemists and molecular pharmacologists, who, together, may pave the way for new EAAT ligands of importance.     

Chemo-enzymatic synthesis of (2S,4R)-2-amino-4-(3-(2,2-diphenylethylamino)-3-oxopropyl)pentanedioic acid: a novel selective inhibitor of human excitatory amino acid transporter subtype 2

In the mammalian central nervous system (CNS), the action of sodium dependent excitatory amino acid transporters (EAATs) is responsible for termination of glutamatergic neurotransmission by reuptake of ( S) -glutamate (Glu) from the synaptic cleft. Five EAAT subtypes have been identified, of which EAAT1-4 are present in the CNS, while EAAT5 is localized exclusively in the retina. In this study, we have used an enantioselective chemo-enzymatic strategy to synthesize 10 new Glu analogues 2a- k ( 2d is exempt) with different functionalities in the 4 R-position and characterized their pharmacological properties at the human EAAT1-3. In particular, one compound, 2k, displayed a significant preference as inhibitor of the EAAT2 subtype over EAAT1,3. The compound also displayed very low affinities toward ionotropic and metabotropic Glu receptors, making it the most selective EAAT2 inhibitor described so far.     

Pharmacology of excitatory amino acid transporters (EAATs and VGLUTs)

L-Glutamate is a major excitatory neurotransmitter in the mammalian central nervous system (CNS). It contributes not only to fast synaptic neurotransmission but also to complex physiological processes like plasticity, learning, and memory. Glutamate is synthesized in the cytoplasm and stored in synaptic vesicles by a proton gradient-dependent uptake system (VGLUTs). Following its exocytotic release, glutamate activates different kinds of glutamate receptors and mediates excitatory neurotransmission. To terminate the action of glutamate and maintain its extracellular concentration below excitotoxic levels, glutamate is quickly removed by Na(+)-dependent glutamate transporters (EAATs). Recently, three vesicular glutamate transporters (VGLUT1-3) and five Na(+)-dependent glutamate transporters (EAAT1-5) were identified. VGLUTs and EAATs are thought to play important roles in neuronal disorders, such as amyotrophic lateral sclerosis, Alzheimer's disease, cerebral ischemia, and Huntington's disease. In this review, the development of new compounds to regulate the function of VGLUTs and EAATs will be described.     

Characterization of novel L-threo-beta-benzyloxyaspartate derivatives, potent blockers of the glutamate transporters

Nontransportable blockers of the glutamate transporters are important tools for investigating mechanisms of synaptic transmission. DL-threo-beta-Benzyloxyaspartate (DL-TBOA) is a potent blocker of all subtypes of the excitatory amino acid transporters (EAATs). We characterized novel L-TBOA analogs possessing a substituent on their respective benzene rings. The analogs significantly inhibited labeled glutamate uptake, the most potent of which was (2S,3S)-3-[3-[4-(trifluoromethyl)benzoylamino]benzyloxy]aspartate (TFB-TBOA). In an uptake assay using cells transiently expressing EAATs, the IC(50) values of TFB-TBOA for EAAT1, EAAT2, and EAAT3 were 22, 17, and 300 nM, respectively. TFB-TBOA was significantly more potent at inhibiting EAAT1 and EAAT2 compared with L-TBOA (IC(50) values for EAAT1-3 were 33, 6.2, and 15 microM, respectively). Electrophysiological analyses revealed that TBOA analogs block the transport-associated currents in all five EAAT subtypes and also block leak currents in EAAT5. The rank order of the analogs for potencies at inhibiting substrate-induced currents was identical to that observed in the uptake assay. However, the kinetics of TFBTBOA differed from the kinetics of L-TBOA, probably because of the strong binding affinity. Notably, TFB-TBOA did not affect other representative neurotransmitter transporters or receptors, including ionotropic and metabotropic glutamate receptors, indicating that it is highly selective for EAATs. Moreover, intracerebroventricular administration of the TBOA analogs induced severe convulsive behaviors in mice, probably because of the accumulation of glutamate. Taken together, these findings indicate that novel TBOA analogs, especially TFB-TBOA, should serve as useful tools for elucidating the physiological roles of the glutamate transporters.     

The substituted aspartate analogue L-beta-threo-benzyl-aspartate preferentially inhibits the neuronal excitatory amino acid transporter EAAT3

The excitatory amino acid transporters (EAATs) play key roles in the regulation of CNS L-glutamate, especially related to synthesis, signal termination, synaptic spillover, and excitotoxic protection. Inhibitors available to delineate EAAT pharmacology and function are essentially limited to those that non-selectively block all EAATs or those that exhibit a substantial preference for EAAT2. Thus, it is difficult to selectively study the other subtypes, particularly EAAT1 and EAAT3. Structure activity studies on a series of beta-substituted aspartate analogues identify L-beta-benzyl-aspartate (L-beta-BA) as among the first blockers that potently and preferentially inhibits the neuronal EAAT3 subtype. Kinetic analysis of D-[(3)H]aspartate uptake into C17.2 cells expressing the hEAATs demonstrate that L-beta-threo-BA is the more potent diastereomer, acts competitively, and exhibits a 10-fold preference for EAAT3 compared to EAAT1 and EAAT2. Electrophysiological recordings of EAAT-mediated currents in Xenopus oocytes identify L-beta-BA as a non-substrate inhibitor. Analyzing L-beta-threo-BA within the context of a novel EAAT2 pharmacophore model suggests: (1) a highly conserved positioning of the electrostatic carboxyl and amino groups; (2) nearby regions that accommodate select structural modifications (cyclopropyl rings, methyl groups, oxygen atoms); and (3) a unique region L-beta-threo-BA occupied by the benzyl moieties of L-TBOA, L-beta-threo-BA and related analogues. It is plausible that the preference of L-beta-threo-BA and L-TBOA for EAAT3 and EAAT2, respectively, could reside in the latter two pharmacophore regions.     

Pharmacological manipulation of glutamate transport

L-Glutamic acid acts as the major excitatory neurotransmitter and, at the same time, represents a potential neurotoxin for the mammalian central nervous system (CNS). The termination of excitatory transmission and the maintenance of physiologic levels of extracellular glutamate, which is necessary to prevent excitotoxicity, are prominently mediated by a family of high-affinity sodium-dependent excitatory amino acid transporters (EAATs). Five subtypes of EAATs have been cloned, possessing distinct pharmacology, localization, sensitivity to transport inhibitors and modulatory mechanisms. Expression and activity of EAATs have been shown to be amenable to fine endogenous and, potentially, pharmacological regulation by substrate itself, growth factors, second messengers, hormones, biological oxidants, inflammatory mediators and pathological conditions. The present review describes basic pharmacological studies, mostly performed on animal models or cell preparations, in order to obtain an updated picture of the known regulatory mechanisms of single EAAT expression and activity. New insight into molecular pathways involved in EAAT regulation will allow pharmacological manipulation of excitatory CNS activity, possibly avoiding adverse effects of glutamate receptor blockade.     

Differentiation of substrate and nonsubstrate inhibitors of the high-affinity, sodium-dependent glutamate transporters

Within the mammalian central nervous system, the efficient removal of L-glutamate from the extracellular space by excitatory amino acid transporters (EAATs) has been postulated to contribute to signal termination, the recycling of transmitter, and the maintenance of L-glutamate at concentrations below those that are excitotoxic. The development of potent and selective inhibitors of the EAATs has contributed greatly to the understanding of the functional roles of these transporters. In the present study, we use a library of conformationally constrained glutamate analogs to address two key issues: the differentiation of substrates from nontransportable inhibitors and the comparison of the pharmacological profile of synaptosomal uptake with those of the individual EAAT clones. We demonstrate that the process of transporter-mediated heteroexchange can be exploited in synaptosomes to rapidly distinguish transportable from nontransportable inhibitors. Using this approach, we demonstrate that 2,4-methanopyrrolidine-2,4-dicarboxylate, cis-1-aminocyclobutane-1,3-dicarboxylate, and L-trans-2, 4-pyrrolidine dicarboxylate act as substrates for the rat forebrain synaptosomal glutamate uptake system. In contrast, L-anti-endo-3, 4-methanopyrrolidine-3,4-dicarboxylate, L-trans-2,3-pyrrolidine dicarboxylate, and dihydrokainate proved to be competitive inhibitors of D-[(3)H]aspartate uptake that exhibited little or no activity as substrates. When these same compounds were characterized for substrate activity by recording currents in voltage-clamped Xenopus laevis oocytes expressing the human transporter clones EAAT1, EAAT2, or EAAT3, it was found that the pharmacological profile of the synaptosomal system exhibited the greatest similarity with the EAAT2 subtype, a transporter believed to be expressed primarily on glial cells.     

Pharmacological characterization of human excitatory amino acid transporters EAAT1, EAAT2 and EAAT3 in a fluorescence-based membrane potential assay

We have expressed the human excitatory amino acid transporters EAAT1, EAAT2 and EAAT3 stably in HEK293 cells and characterized the transporters pharmacologically in a conventional [(3) H]-d-aspartate uptake assay and in a fluorescence-based membrane potential assay, the FLIPR Membrane Potential (FMP) assay. The K(m) and K(i) values obtained for 12 standard EAAT ligands at EAAT1, EAAT2 and EAAT3 in the FMP assay correlated well with the K(i) values obtained in the [(3) H]-d-aspartate assay (r(2) values of 0.92, 0.92, and 0.95, respectively). Furthermore, the pharmacological characteristics of the cell lines in the FMP assay were in good agreement with previous findings in electrophysiology studies of the transporters. The FMP assay was capable of distinguishing between substrates and non-substrate inhibitors and to discriminate between "full" and "partial" substrates at the transporters. Taking advantage of the prolific nature of the FMP assay, interactions of the EAATs with substrates and inhibitors were studied in some detail. This is the first report of a high throughput screening assay for EAATs. We propose that the assay will be of great use in future studies of the transporters. Although conventional electrophysiology set-ups might be superior in terms of studying sophisticated kinetic aspects of the uptake process, the FMP assay enables the collection of considerable amounts of highly reproducible data with relatively little labor. Furthermore, considering that the number of EAAT ligands presently available is limited, and that almost all of these are characterized by low potency and a low degree of subtype selectivity, future screening of compound libraries at the EAAT-cell lines in the FMP assay could help identify structurally and pharmacologically novel ligands for the transporters.     

Expression of GFP-tagged neuronal glutamate transporters in cerebellar Purkinje neurons

Of the five excitatory amino acid transporters (EAATs) identified, two genes are expressed by neurons (EAAT3 and EAAT4) and give rise to transporters confined to neuronal cell bodies and dendrites. At an ultrastructural level, EAAT3 and EAAT4 proteins are clustered at the edges of postsynaptic densities of excitatory synapses. This pattern of localization suggests that postsynaptic EAATs may help to limit spillover of glutamate from excitatory synapses. In an effort to study transporter localization in living neurons and ultimately to manipulate uptake at intact synapses, we have developed viral reagents encoding neuronal EAATs tagged with GFP. We demonstrate that these fusion proteins are capable of Na(+)-dependent glutamate uptake, that they generate ionic conductances indistinguishable from their wild-type counterparts, and that GFP does not alter their glutamate dose-dependence. Two-photon microscopy was used to examine fusion protein expression in Purkinje neurons in acute cerebellar slices. Both EAAT3-GFP and EAAT4-GFP were observed at high levels in the dendritic spines of transfected Purkinje neurons. These findings indicate that functional EAAT fusion proteins can be synthesized and appropriately trafficked to postsynaptic compartments. Furthermore, they validate a powerful system for looking at EAAT function in situ.     

Characterization of the tritium-labeled analog of L-threo-beta-benzyloxyaspartate binding to glutamate transporters

L-Glutamate is the major excitatory neurotransmitter in the mammalian central nervous system. Termination of glutamate receptor activation and maintenance of low extracellular glutamate concentrations are primarily achieved by glutamate transporters (excitatory amino acid transporters 1-5, EAATs1-5) located on both the nerve endings and the surrounding glial cells. To identify the physiological roles of each subtype, subtype-selective EAAT ligands are required. In this study, we developed a binding assay system to characterize EAAT ligands for all EAAT subtypes. We recently synthesized novel analogs of threo-beta-benzyloxyaspartate (TBOA) and reported that they blocked glutamate uptake by EAATs 1-5 much more potently than TBOA. The strong inhibitory activity of the TBOA analogs suggested that they would be suitable to use as radioisotope-labeled ligands, and we therefore synthesized a tritiated derivative of (2S,3S)-3-{3-[4-ethylbenzoylamino]benzyloxy}aspartate ([3H]ETB-TBOA). [3H]ETB-TBOA showed significant high-affinity specific binding to EAAT-transfected COS-1 cell membranes with each EAAT subtype. The Hill coefficient for the Na+-dependence of [3H]ETB-TBOA binding revealed a single class of noncooperative binding sites for Na+, suggesting that Na+ binding in the ligand binding step is different from Na+ binding in the substrate uptake process. The binding was displaced by known substrates and blockers. The rank order of inhibition by these compounds was consistent with glutamate uptake assay results reported previously. Thus, the [3H]ETB-TBOA binding assay will be useful to screen novel EAAT ligands for all EAAT subtypes.     

Roles and regulation of glutamate transporters in the central nervous system

1. Glutamate transporters (also known as excitatory amino acid transporters or EAAT) are solely responsible for the removal of the excitatory neurotransmitter l-glutamate (Glu) from the extracellular space and, thus, permit normal transmission, as well as preventing cell death due to the excessive activation of Glu receptors. 2. Five subtypes of glutamate transporter (EAAT1-5) exist, possessing distinct pharmacology, cellular localization and modulatory mechanisms. 3. Experimental inhibition of EAAT activity in vitro and in vivo results in increased extracellular concentrations of Glu and in neuronal death via excitotoxicity, highlighting the importance of EAAT in normal excitatory neurotransmission. 4. Dysfunction of EAAT may contribute to the pathology of both acute neuronal injury and chronic neurodegenerative conditions, so correction of EAAT function under these conditions may provide a valuable therapeutic strategy. 5. The present review describes basic pharmacological studies that allow new insights into EAAT function and suggest possible strategies for the therapeutic modulation of EAAT.     

Excitatory amino acid transporters as emerging targets for central nervous system therapeutics

In the mammalian central nervous system (CNS) the excitatory amino acid transporter (EAAT) family of proteins are responsible for the high-affinity sodium-dependent uptake of glutamate into both astroglial cells and neurones. Normal EAAT function is required both for the efficient termination of glutamatergic neurotransmission and for the maintenance of low extracellular glutamate concentrations, thereby preventing glutamate excitotoxicity. It is widely believed that a dysfunction of glutamate transmission participates in the aetiology of a number of neurodegenerative and neuropsychiatric disorders and diseases. This review introduces the EAATs as a new family of emerging therapeutic targets for CNS disorders by virtue of their central role in maintaining glutamate homeostasis. We examine recent findings on the modulation and regulation of EAATs and review the changes in both EAAT function and expression which have been described in a number of neuropathological conditions.     

Glutamate-based therapeutic approaches: targeting the glutamate transport system

Although the ionotropic and metabotropic receptors for synaptically released glutamate have been extensively mined in the pursuit of novel therapeutic agents for a diverse array of central nervous system disorders, pursuit of the transport proteins--or excitatory amino acid transporters (EAATs)--toward a similar end has been a road much less travelled. Recent progress has seen the use of cloned EAAT subtypes to develop transporter inhibitors with improved subtype selectivity, providing important tools for elucidating the precise contribution of each transporter subtype to the regulation of extracellular glutamate homeostasis. In addition, momentum has been gained with the discovery of compounds capable of upregulating the activity of the predominant forebrain glutamate transporter, EAAT2.     

Glutamate transporters as drug targets

The L-glutamate (Glu) has been hypothesized as an excitatory amino acid neurotransmitter in the mammalian central nervous system after successful cloning and identification of a number of genes encoding signaling machineries required for the neurocrine at synapses in the brain. These include excitatory amino acid transporters (EAATs) for signal termination and vesicular Glu transporters (VGLUTs) for signal output through exocytotic release, in addition to Glu receptors (GluRs) for signal input. These Glu signaling molecules not only play key roles in mechanisms associated with synaptic plasticity such as learning and memory, but also participate in the etiology and pathology of different neuropsychiatric disorders and neuronal cell death seen in various neurodegenerative diseases. Of the aforementioned Glu signaling molecules, EAATs are essential for the termination of signal transmission mediated by Glu as well as for the prevention of neurotoxicity mediated by this endogenous excitotoxin, while VGLUTs are crucial for the storage of Glu in synaptic vesicles to suffice for the definition of a glutamatergic phenotype. Many early desperate efforts were devoted to the search and development of novel compounds with a therapeutic window toward GluRs, while relatively little attention was paid to either EAATs or VGLUTs in this aspect. In this review, therefore, we will summarize the classification and functionality of EAATs and VGLUTs with a focus on their possibilities as potential therapeutic targets for different neurodegenerative and neuropsychiatric disorders related to malfunction of Glu signaling in human beings.     

Substrate-induced modulation of glutamate uptake in human platelets

In the central nervous system (CNS), glutamate rapidly upregulates the activities of different excitatory amino-acid transporter subtypes (EAATs) in order to help protect neurons from excitotoxicity. Since human platelets display a specific sodium-dependent glutamate uptake activity, and express the three major glutamate transporters, which may be affected in neurological disorders, we investigated whether platelets are subject to substrate-induced modulation as described for CNS. A time- and dose-dependent upregulation of [3H]-glutamate uptake (up to two-fold) was observed in platelets preincubated with glutamate. There was an increase in maximal velocity rate without affinity changes. Glutamate receptor agonists and antagonists did not modulate this upregulation and preincubation with glutamate analogues failed to mimic the glutamate effect. Only aspartate preincubation increased the uptake, albeit approximately 35% less with respect to glutamate. The effect of glutamate preincubation on the expression of the three major transporters was studied by Western blotting, showing an increase of approximately 70% in EAAT1 immunoreactivity that was completely blocked by cycloheximide (CEM). However, L-serine-O-sulphate, at a concentration (200 microM) known to block EAAT1/3 selectively, did not completely inhibit the effect of glutamate stimulation, indicating the possible involvement of EAAT2. In fact, glutamate stimulation was completely abolished only when, following CEM pre-incubation, the experiment was run in the presence of the selective EAAT2 inhibitor dihydrokainic acid. Since surface biotinylation experiments failed to show evidence of EAAT2 translocation, our results suggest the existence of a different way of regulating EAAT2 activity. These findings indicate that human platelets display a substrate-dependent modulation of glutamate uptake mediated by different molecular mechanisms and confirm that ex vivo platelets are a reliable model to investigate the dysfunction of glutamate uptake regulation in patients affected by neurological disorders.     

Functional and morphological characterization of glutamate transporters in the rat locus coeruleus

Background and Purpose

Excitatory amino acid transporters (EAATs) in the CNS contribute to the clearance of glutamate released during neurotransmission. The aim of this study was to explore the role of EAATs in the regulation of locus coeruleus (LC) neurons by glutamate.

Experimental Approach

We measured the effect of different EAAT subtype inhibitors/enhancers on glutamate- and KCl-induced activation of LC neurons in rat slices. EAAT2–3 expression in the LC was also characterized by immunohistochemistry.

Key Results

The EAAT2–5 inhibitor DL-threo-β-benzyloxaspartic acid (100 μM), but not the EAAT2, 4, 5 inhibitor L-trans-pyrrolidine-2,4-dicarboxylic acid (100 μM) or the EAAT2 inhibitor dihydrokainic acid (DHK; 100 μM), enhanced the glutamate- and KCl-induced activation of the firing rate of LC neurons. These effects were blocked by ionotropic, but not metabotrobic, glutamate receptor antagonists. DHK (100 μM) was the only EAAT inhibitor that increased the spontaneous firing rate of LC cells, an effect that was due to inhibition of EAAT2 and subsequent AMPA receptor activation. Chronic treatment with ceftriaxone (200 mg·kg⁻¹ i.p., once daily, 7 days), an EAAT2 expression enhancer, increased the actions of glutamate and DHK, suggesting a functional impact of EAAT2 up-regulation on the glutamatergic system. Immuhistochemical data revealed the presence of EAAT2 and EAAT3 surrounding noradrenergic neurons and EAAT2 on glial cells in the LC.

Conclusions and Implications

These results remark the importance of EAAT2 and EAAT3 in the regulation of rat LC by glutamate. Neuronal EAAT3 would be responsible for terminating the action of synaptically released glutamate, whereas glial EAAT2 would regulate tonic glutamate concentrations in this nucleus.     

Roles of ATP receptors in the regulation of various functions in spinal microglia

It is shown that glial cells have a pivotal influence on the formation of neuronal network in central nerve system. Moreover, spinal microglia has some important roles in the development and progression of various neurological disorders. Therefore, it is possible that modulation of microglial activity may be sufficient to alleviate those harmful responses. ATP is one of signaling molecules in the spinal cord, and involved in regulation of several microglial functions through the binding of P2X and P2Y receptors. Thus, I focused on the ATP-mediated regulation mechanisms for the two important proteins, which are p38 MAP kinase and excitatory amino acid transporters (EAATs), in cultured spinal microglia. Mounting evidence indicates that p38 in spinal microglia has crucial roles in some neurological diseases. Furthermore, it is recently suggested that microglial EAATs might participate in the homeostasis of glutamate in synapses. This review summarizes our finding regarding the involvement of P2Y receptors and β-adrenergic receptors in the regulation of p38 phosphorylation, and the mechanism of P2X7 receptor-mediated downregulation of EAATs function.     

Differential cocaine-induced modulation of glutamate and dopamine transporters after contingent and non-contingent administration

Although dopamine and glutamate transmission has been implicated in cocaine dependence, the effects of the extinction of cocaine self-administration on protein transporters in both of these neurotransmitter systems remain unknown. We have used a yoked-box procedure to simultaneously test rats in triads, one rat that actively self-administered cocaine (CONT), while the other two received yoked injections of either cocaine (NON-CONT) or saline (SALINE). The brains in each triad were removed and processed for quantitative autoradiography immediately after the last session of cocaine self-administration (Day 0), or after 1, 5, or 10 days of extinction, and excitatory amino acid transporters (EAATs) and dopamine transporter (DAT) binding was examined. When compared to NON-CONT and SALINE animals, binding of radioligand to EAATs was significantly lower in the hippocampal CA1 field and the cerebellar cortex of CONT rats on Day 0, although it was significantly higher after 1 day of extinction in the infralimbic cortex. No differences in EAAT binding were observed after 5 or 10 days of extinction in any of the brain regions analyzed. In contrast and at all the time points of extinction, binding to DAT was significantly enhanced in CONT animals when compared to SALINE and NON-CONT rats in different forebrain and mesencephalic regions, including the nucleus accumbens, ventral tegmental area or caudate putamen. These results suggest that changes in protein transporter binding after cocaine self-administration and extinction are transient for EAAT while they are more enduring for DAT, and that they depend on the type of access to cocaine.