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.     

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.     

Neuronal high-affinity sodium-dependent glutamate transporters (EAATs): targets for the development of novel therapeutics against neurodegenerative diseases

L-Glutamate is the major excitatory neurotransmitter in mammalian central nervous system, and excitatory amino acid transporters (EAATs) are essential for terminating synaptic excitation and for maintaining extracellular glutamate concentration below toxic levels. Although the structure of these channel-like proteins has not been yet reported, their membrane topology has been hypothesised based on biochemical and protein sequence analyses. In the case of an inadequate clearance from synaptic cleft and from the extrasynaptic space, glutamate behaves as a potent neurotoxin, and it may be related to several neurodegenerative pathologies including epilepsy, ischemia, amyotrophic lateral sclerosis, and Alzheimer disease. The recent boom of glutamate is demonstrated by the enormous amount of publications dealing with the function of glutamate, with its role on modulation of synaptic transmission throughout the brain, mainly focusing: i). on the structure of its receptors, ii). on molecular biology and pharmacology of Glu transporters, and iii). on the role of glutamate uptake and reversal uptake in several neuropathologies. This review will deal with the recent and most interesting published results on Glu transporters membrane topology, Glu transporters physiopathological role and Glu transporters medicinal chemistry, highlighting the guidelines for the development of potential neuroprotective agents targeting neuronal high-affinity sodium-dependent glutamate transporters.     

Neuropsychiatric disorders and GABA]

In the mammalian central nervous system (CNS), the inhibitory GABAergic system is composed of different signaling molecules such as glutamate decaroxylase, vesicular GABA transporters, GABA receptors, GABA transporters and GABA transaminase. A prevailing view is that the balance between excitatory signaling mediated by glutamate and inhibitory signaling mediated by GABA plays a pivotal role in mechanisms underlying the modulation and maintenance of a variety of neural functions. Therefore, abnormalities in a GABAergic signaling molecule would lead to a crisis of severe symptoms relevant to a number of neuropsychiatric disorders. These include epilepsy, depression, schizophrenia, stiff-person syndrome, drug addiction and so on. In this review article, we will summarize recent studies on the relationship between the malfunction of GABAergic signaling molecules and the etiology of these neuropsychiatric disorders. We will also refer to novel strategies on GABAergic signaling molecules other than GABA receptors for therapeutic usefulness in the future.     

Pharmacological topics of bone metabolism: glutamate as a signal mediator in bone

The view that L-glutamate (Glu) is an excitatory amino acid neurotransmitter in the mammalian central nervous system is prevailing on the basis of successful cloning of a number of genes encoding different signaling molecules, such as Glu receptors for the signal input, Glu transporters for the signal termination and vesicular Glu transporters for the signal output through exocytotic release. Little attention has been paid to an extracellular transmitter role of Glu in peripheral neuronal and non-neuronal tissues, by contrast, whereas recent molecular biological and pharmacological analyses including ours give rise to a novel function for Glu as an autocrine and/or paracrine signal mediator in bone comprised of osteoblasts, osteoclasts and osteocytes, in addition to other peripheral tissues including pancreas, adrenal and pituitary glands. Emerging evidence suggests that Glu could play a dual role in mechanisms underlying the maintenance of cellular homeostasis as an excitatory neurotransmitter in the central nervous system and as an extracellular signal mediator in peripheral autocrine and/or paracrine tissues. In this review, therefore, we would outline the possible signaling system for Glu to play a role as an extracellular signal mediator in mechanisms underlying maintenance of the cellular homeostasis in bone.     

Metabotropic glutamate receptors as drug targets

L-glutamate (Glu), the main excitatory amino acid neurotransmitter in the mammalian central nervous system, is involved in many physiological functions, including learning and memory, but also in toxic phenomena occurring in numerous degenerative or neurological diseases. These functions mainly result from its interaction with Glu receptors (GluRs). The broad spectrum of roles played by glutamate derived from the large number of membrane receptors, which are currently classified in two main categories, ionotropic (iGluRs) and metabotropic (mGluRs) receptors. The iGluRs are ion channels, permeant to Na(+) (Ca(2+)) while the mGluRs belongs to the superfamily of G-protein coupled receptors (GPCRs). Despite continuous efforts over more than two decades, the use of iGluR agonists or antagonists to improve or inhibit excitatory transmission in pathological states still remains a major challenge, though the discovery and development of recent molecules may prove it worthwhile. This probably results form the vital role of fast excitatory transmission in many fundamental physiological functions. Since the discovery of mGluRs, hope has emerged. Indeed, mGluRs are mainly involved in the regulation of fast excitatory transmission. Consequently, it was logically thought that modulating mGluRs with agonists or antagonists might lead to more subtle regulation of fast excitatory transmission than by directly blocking iGluRs. As a result of intensive investigation, new drugs permitting to discriminate between these receptors have emerged. Moreover, a new class of molecules acting as negative or positive allosteric modulators or mGluRs is now available and appears to be promising. In the following, we will review the classification of mGluRs and the functions in which mGluRs are involved. We will focus on their potential as therapeutic targets for improving numerous physiological functions and for different neurodegenerative and neuropsychiatric disorders, which are related to malfunction of Glu signaling in human beings.     

Glutamate signaling system in bone

L-glutamate (Glu) has been thought to be an excitatory amino acid neurotransmitter in the mammalian central nervous system (CNS). The hypothesis is supported by successful cloning of a number of genes encoding different signaling molecules, such as Glu receptors for signal input, Glu transporters for signal termination, and vesicular Glu transporters for signal output through exocytotic release. Limited information is available in the literature with regard to an extracellular transmitter role of Glu in peripheral neuronal and non-neuronal tissues, whereas recent molecular biological analyses including ours give rise to a novel function for Glu as an autocrine and/or paracrine factor in bone comprised of osteoblasts, osteoclasts, and osteocytes, in addition to other peripheral tissues including pancreas, adrenal, and pituitary glands. Emerging evidence suggests that Glu could play a dual role in mechanisms underlying maintenance of cellular homeostasis as an excitatory neurotransmitter in the CNS and as an extracellular signal mediator in peripheral autocrine and/or paracrine tissues. In this review, therefore, we summarized the possible signaling by Glu as an extracellular signal mediator in mechanisms underlying maintenance of cellular homeostasis with a focus on bone tissues.     

Possible involvement of glutamatergic signaling machineries in pathophysiology of rheumatoid arthritis

The prevailing view is that L-glutamate (Glu) functions as an excitatory amino acid neurotransmitter through a number of molecular machineries required for the neurocrine signaling at synapses in the brain. These include Glu receptors for signal input, Glu transporters for signal termination, and vesicular Glu transporters for signal output through exocytotic release. Although relatively little attention has been paid to the functional expression of these molecules required for glutamatergic signaling in peripheral tissues, recent molecular biological analyses including ours give rise to a novel function for Glu as an extracellular signal mediator in the autocrine and/or paracrine system in several peripheral and non-neuronal tissues, including bone and cartilage. In particular, a drastic increase is demonstrated in the endogenous levels of both Glu and aspartate in the synovial fluid with intimate relevance to increased edema and sensitization to thermal hyperalgesia in experimental arthritis models. However, to date, there is only limited information about the physiological and pathological significance of glutamatergic signaling machineries expressed by articular synovial tissues. In this review, we have outlined the role of Glu in synovial fibroblasts in addition to the possible involvement of glutamatergic signaling machineries in the pathogenesis of joint diseases such as rheumatoid arthritis.     

Parawixin1: a spider toxin opening new avenues for glutamate transporter pharmacology

Glutamate is the major excitatory neurotransmitter in the mammalian central nervous system. After release from glutamatergic nerve terminals, glial and neuronal glutamate transporters remove glutamate from the synaptic cleft to terminate synaptic transmission and to prevent neuronal damage by excessive glutamate receptor activation. In this issue of Molecular Pharmacology, Fontana et al. (p. 1228) report on the action of a venom compound, Parawixin1, on excitatory amino acid transporters (EAATs). They demonstrate that this agent selectively affects a glial glutamate transporter, EAAT2, by specifically increasing one particular step of the glutamate uptake cycle. Disturbed glutamate homeostasis seems to be a pathogenetic factor in several neurodegenerative disorders. Because EAAT2 is a key player in determining the extracellular glutamate concentration in the mammalian brain, drugs targeting this protein could prevent glutamate excitotoxicity without blocking glutamatergic transmission. Its specificity and selectivity makes Parawixin1 a perfect starting point to design small molecules for the treatment of pathological conditions caused by alterations of glutamate homeostasis.     

Transporters for L-glutamate: an update on their molecular pharmacology and pathological involvement

L-Glutamate (Glu) is the major excitatory neurotransmitter in the mammalian CNS and five types of high-affinity Glu transporters (EAAT1-5) have been identified. The transporters EAAT1 and EAAT2 in glial cells are responsible for the majority of Glu uptake while neuronal EAATs appear to have specialized roles at particular types of synapses. Dysfunction of EAATs is specifically implicated in the pathology of neurodegenerative conditions such as amyotrophic lateral sclerosis, epilepsy, Huntington's disease, Alzheimer's disease and ischemic stroke injury, and thus treatments that can modulate EAAT function may prove beneficial in these conditions. Recent advances have been made in our understanding of the regulation of EAATs, including their trafficking, splicing and post-translational modification. This article summarises some recent developments that improve our understanding of the roles and regulation of EAATs.     

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.     

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.     

Role for GABA and Glu plasma membrane transporters in the interplay of inhibitory and excitatory neurotransmission

Neurotransmitter plasma membrane transporters do have much more to perform than simply terminating synaptic transmission and replenishing neurotransmitter pools. Findings in the past decade have evidenced their function in maintaining physiological synaptic excitability, and their actions in critical or pathological conditions, also. Conclusively these findings indicated a previously unrecognized role for neurotransmitter plasma membrane transporters in both, synaptic and nonsynaptic signaling. Major inhibitory and excitatory neurotransmitters within the brain, GABA and Glu, have long been considered to operate through independent systems (GABAergic or Gluergic), each of them characterized by its own localization, function and dedicated GABAergic or Gluergic cell phenotypes. Recent advances, however, have challenged this long-standing paradigm. Localization of GABA in Gluergic terminals and Glu in GABAergic cells were reported. Specific plasma membrane transporters for GABA and Glu are also co-localized in different brain areas. Although, their role in regulating each other's signal is still far from being understood, emerging lines of evidence on interplaying GABAergic and Gluergic processes through plasma membrane transporters opens up a new avenue in the field of more specific therapeutic intervention.     

Can selective ligands for glutamate binding proteins be rationally designed?

A major neurotransmitter, L-Glutamate must be stored, transported and received, and these processes are mediated by proteins that bind this simple yet essential amino acid. Detailed evidence continues to emerge on the structure of Glu binding proteins, which includes both receptors and transporters. It appears that receptors and transporters bind to Glu in different conformations, which may present a pharmacological opportunity. This review will compare and contrast information available on Glu Receptors (AMPA, NMDA, KA and mGlu), excitatory amino acid transporters (EAATs), the system Xc- transporter (XCT) and the vesicular Glutamate transporter (GVT). The cross-reactivity of ligands which have been previously used to characterize the glutamate binding proteins with system Xc- raises some fundamental interpretational issues regarding the mechanisms through which these analogues produce CNS damage. Although at one time it was thought that unraveling selectivity among glutamate binding proteins was an intractable problem, recently the NMDA antagonist (memantine) has been approved for general medical practice for treatment of Alzheimer's disease. Two other agents are in advanced clinical trials: an Ampakine for potential improvement of cognitive disorders, and a selective mGlu agonist for treatment of anxiety. The prospects for unraveling cross-reactivity will be weighed in light of a critical comparison of the glutamate binding protein targets.     

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.     

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.     

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.     

兴奋性氨基酸转运体研究进展

兴奋性氨基酸转运体 (EAAT)位于突触前膜、突触囊泡和神经胶质细胞膜上。它们对于兴奋性氨基酸的再循环 ,兴奋性信号的终止以及保护神经细胞免受兴奋性毒性损害具有特别重要的意义。本文介绍EAAT研究进展     

Zn2+ modulation of neurotransmitter transporters

Neurotransmitter transporters located at the presynaptic or glial cell membrane are responsible for the stringent and rapid clearance of the transmitter from the synapse, and hence they terminate signaling and control the duration of synaptic inputs in the brain. Two distinct families of neurotransmitter transporters have been identified based on sequence homology: (1) the neurotransmitter sodium symporter family (NSS), which includes the Na+/C1(-)-dependent transporters for dopamine, norepinephrine, and serotonin; and (2) the dicarboxylate/amino acid cation symporter family (DAACS), which includes the Na(+)-dependent glutamate transporters (excitatory amino acid transporters; EAAT). In this chapter, we describe how the identification of endogenous Zn2(+)-binding sites, as well as engineering of artificial Zn2(+)-binding sites both in the Na+/Cl(-)-dependent transporters and in the EAATs, have proved to be an important tool for studying the molecular function of these proteins. We also interpret the current available data on Zn2(+)-binding sites in the context of the recently published crystal structures. Moreover, we review how the identification of endogenous Zn2(+)-binding sites has indirectly suggested the possibility that several of the transporters are modulated by Zn2+ in vivo, and thus that Zn2+ can play a role as a neuromodulator by affecting the function of neurotransmitter transporters.