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.     

Regulation and dysregulation of glutamate transporters

Glutamate is the primary excitatory neurotransmitter in the central nervous system. During synaptic activity, glutamate is released into the synaptic cleft and binds to glutamate receptors on the pre- and postsynaptic membrane as well as on neighboring astrocytes in order to start a number of intracellular signaling cascades. To allow for an efficient signaling to occur, glutamate levels in the synaptic cleft have to be maintained at very low levels. This process is regulated by glutamate transporters, which remove excess extracellular glutamate via a sodium-potassium coupled uptake mechanism. When extracellular glutamate levels rise to about normal, glutamate overactivates glutamate receptors, triggering a multitude of intracellular events in the postsynaptic neuron, which ultimately results in neuronal cell death. This phenomenon is known as excitotoxicity and is the underlying mechanisms of a number of neurodegenerative diseases. A dysfunction of the glutamate transporter is thought to contribute to cell death during excitotoxicity. Therefore, efforts have been made to understand the regulation of glutamate transporter function. Transporter activity can be regulated in different ways, including through gene expression, transporter protein targeting and trafficking and through posttranslational modifications of the transporter protein. The identification of these mechanisms has helped to understand the role of glutamate transporters during pathology and will aid in the development of therapeutic strategies with the transporter as a desirable target.     

Lessons from the knocked-out glycine transporters

Glycine has multiple neurotransmitter functions in the central nervous system (CNS). In the spinal cord and brainstem of vertebrates, it serves as a major inhibitory neurotransmitter. In addition, it participates in excitatory neurotransmission by modulating the activity of the N-methyl-D-aspartate (NMDA) subtype of glutamate receptors. The extracellular concentrations of glycine are regulated by Na+/Cl(-)-dependent glycine transporters (GlyTs), which are expressed in neurons and adjacent glial cells. Considerable progress has been made recently towards elucidating the in vivo roles of GlyTs in the CNS. The generation and analysis of animals carrying targeted disruptions of GlyT genes (GlyT knockout mice) have allowed investigators to examine the different contributions of individual GlyT subtypes to synaptic transmission. In addition, they have provided animal models for two hereditary human diseases, glycine encephalopathy and hyperekplexia. Selective GlyT inhibitors have been shown to modulate neurotransmission and might constitute promising therapeutic tools for the treatment of psychiatric and neurological disorders such as schizophrenia and pain. Therefore, pharmacological and genetic studies indicate that GlyTs are key regulators of both glycinergic inhibitory and glutamatergic excitatory neurotransmission. This chapter describes our present understanding of the functions of GlyTs and their involvement in the fine-tuning of neuronal communication.     

Glutamate receptors: variation in structure-function coupling

Fast excitatory synaptic transmission in the CNS relies almost entirely on the neurotransmitter glutamate and its family of ion channel receptors. An appreciation of the coupling between agonist binding and channel opening has advanced rapidly during the past five years, largely as a result of new structural information about the agonist-binding site. Recent studies suggest that despite many structural similarities different family members use different mechanisms to translate agonist binding into channel opening.     

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.     

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.     

MOLECULAR PHARMACOLOGY AND PHYSIOLOGY OF GLUTAMATE TRANSPORTERS IN THE CENTRAL NERVOUS SYSTEM

1. Glutamate is the predominant excitatory neurotransmitter in the brain, but it is also a potent neurotoxin. Following release of glutamate from presynaptic vesicles into the synapse and activation of a variety of ionotropic and metabotropic glutamate receptors, glutamate is removed from the synapse. This is achieved through active uptake of glutamate by transporters located pre- and also post-synaptically or, alternatively, glutamate can diffuse out of the synapse and be taken up by transporters located on the cell surface of glial cells. 2. Complementary DNA encoding a number of glutamate transporters have recently been cloned and form a family of structurally related membrane proteins with a high degree of amino acid sequence conservation. Expression of the cloned glutamate transporters in various cell types has aided in the characterization of the functional properties of the different transporter subtypes. 3. Glutamate transport is coupled to sodium, potassium and pH gradients across the cell membrane creating an electrogenic process. This allows transport to be measured using electrophysiological techniques, which has greatly aided in understanding some of the basic mechanisms of the transport process and has also allowed a detailed understanding of the molecular pharmacology of the different transporter subtypes. 4. In the present review I shall discuss some of the recent advances in understanding the molecular basis for glutamate transporter function and then highlight some of the unanswered questions concerning the physiological roles of these proteins and suggest possible strategies for pharmacological manipulation of transporters for the treatment of neurological disorders.     

Sulfated steroids as endogenous neuromodulators

Central nervous system function is critically dependent upon an exquisitely tuned balance between excitatory synaptic transmission, mediated primarily by glutamate, and inhibitory synaptic transmission, mediated primarily by GABA. Modulation of either excitation or inhibition would be expected to result in altered functionality of finely tuned synaptic pathways and global neural systems, leading to altered nervous system function. Administration of positive or negative modulators of ligand-gated ion channels has been used extensively and successfully in CNS therapeutics, particularly for the induction of sedation and treatment of anxiety, seizures, insomnia, and pain. Excessive activation of excitatory glutamate receptors, such as in cerebral ischemia, can result in neuronal damage via excitotoxic mechanisms. The discovery that neuroactive steroids exert rapid, direct effects upon the function of both excitatory and inhibitory neurotransmitter receptors has raised the possibility that endogenous neurosteroids may play a regulatory role in synaptic transmission by modulating the balance between excitatory and inhibitory neurotransmission. The sites to which neuroactive steroids bind may also serve as targets for the discovery of therapeutic neuromodulators.     

Etomidate reduces glutamate uptake in rat cultured glial cells: involvement of PKA

Background and purpose:

Glutamate is the main excitatory neurotransmitter in the vertebrate CNS. Removal of the transmitter from the synaptic cleft by glial and neuronal glutamate transporters (GLTs) has an important function in terminating glutamatergic neurotransmission and neurological disorders. Five distinct excitatory amino-acid transporters have been characterized, among which the glial transporters excitatory amino-acid transporter 1 (EAAT1) (glutamate aspartate transporter) and EAAT2 (GLT1) are most important for the removal of extracellular glutamate. The purpose of this study was to describe the effect of the commonly used anaesthetic etomidate on glutamate uptake in cultures of glial cells.

Experimental approach:

The activity of the transporters was determined electrophysiologically using the whole cell configuration of the patch-clamp recording technique.

Key results:

Glutamate uptake was suppressed by etomidate (3–100 μM) in a time- and concentration-dependent manner with a half-maximum effect occurring at 2.4±0.6 μM. Maximum inhibition was approximately 50% with respect to the control. Etomidate led to a significant decrease of V_max whereas the K_m of the transporter was unaffected. In all cases, suppression of glutamate uptake was reversible within a few minutes upon washout. Furthermore, both GF 109203X, a nonselective inhibitor of PKs, and H89, a selective blocker of PKA, completely abolished the inhibitory effect of etomidate.

Conclusion and implications:

Inhibition of glutamate uptake by etomidate at clinically relevant concentrations may affect glutamatergic neurotransmission by increasing the glutamate concentration in the synaptic cleft and may compromise patients suffering from acute or chronic neurological disorders such as CNS trauma or epilepsy.     

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.     

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.     

谷氨酸转运体靶点药物的抗脑缺血作用研究进展

兴奋性氨基酸毒性是脑缺血损伤的主要机制之一。缺血期间谷氨酸的大量累积会导致神经元细胞、星形胶质细胞等神经细胞发生兴奋性毒性损伤,因此对缺血期间谷氨酸水平的调控一直是脑缺血防治药物研究的重点。近年来研究表明,通过上调星形胶质细胞上谷氨酸转运体GLAST(EAAT1)和GLT-1(EAAT2)的表达或活性,增加缺血时谷氨酸的摄取,维持突触间隙内谷氨酸的正常浓度,从而降低兴奋性毒性,减轻缺血性脑损伤。一些化合物如β-内酰胺类抗生素、尿酸、甲状腺激素、雌激素、山楂酸等已在体内或体外实验中被证实对谷氨酸转运体的调节作用,对抗谷氨酸毒性,发挥神经保护作用。研究和开发以星形胶质细胞谷氨酸转运体为作用靶点的药物,为缺血性脑损伤的预防和治疗提供了一条新的途径。     

Regulation of vesicular monoamine and glutamate transporters by vesicle-associated trimeric G proteins: new jobs for long-known signal transduction molecules

Neurotransmitters of neurons and neuroendocrine cells are concentrated first in the cytosol and then in either small synaptic vesicles ofpresynaptic terminals or in secretory vesicles by the activity of specific transporters of the plasma and the vesicular membrane, respectively. In the central nervous system the postsynaptic response depends--amongst other parameters-on the amount of neurotransmitter stored in a given vesicle. Neurotransmitter packets (quanta) vary over a wide range which may be also due to a regulation of vesicular neurotransmitter filling. Vesicular filling is regulated by the availability of transmitter molecules in the cytoplasm, the amount of transporter molecules and an electrochemical proton-mediated gradient over the vesicular membrane. In addition, it is modulated by vesicle-associated heterotrimeric G proteins, Galphao2 and Galphaq. Galphao2 and Galphaq regulate vesicular monoamine transporter (VMAT) activities in brain and platelets, respectively. Galphao2 also regulates vesicular glutamate transporter (VGLUT) activity by changing its chloride dependence. It appears that the vesicular content activates the G protein, suggesting a signal transduction from the luminal site which might be mediated by a vesicular G protein-coupled receptor or as an alternative possibility by the transporter itself. Thus, G proteins control transmitter storage and thereby probablylink the regulation of the vesicular content to intracellular signal cascades.     

Neurotransmitter transporters: molecular function of important drug targets

The concentration of neurotransmitters in the extracellular space is tightly controlled by distinct classes of membrane transport proteins. This review focuses on the molecular function of two major classes of neurotransmitter transporter that are present in the cell membrane of neurons and/or glial cells: the solute carrier (SLC)1 transporter family, which includes the transporters that mediate the Na(+)-dependent uptake of glutamate, and the SLC6 transporter family, which includes the transporters that mediate the Na(+)-dependent uptake of dopamine, 5-HT, norepinephrine, glycine and GABA. Recent research has provided substantial insight into the structure and function of these transporters. In particular, the recent crystallizations of bacterial homologs are of the utmost importance, enabling the first reliable structural models of the mammalian neurotransmitter transporters to be generated. These models should be an important tool for developing specific drugs that, through selective interaction with transporters, could improve the treatment of serious neurological and psychiatric disorders.     

脑缺血时谷氨酸释放机制

谷氨酸是中枢神经系统主要的兴奋性神经递质,在脑缺血造成的神经元损伤过程中发挥重要作用。脑缺血时多种机制参与了谷氨酸释放的的调节,如Ca2+依赖性的出胞式释放、谷氨酸转运体调节的释放、水肿诱发的释放和受体调节的释放等。本文根据现有的文献资料,综述了脑缺血时谷氨酸释放机制。     

Glycine transporter isoforms in the mammalian central nervous system: structures, functions and therapeutic promises

The amino acid glycine (Gly) serves as a neurotransmitter at excitatory and inhibitory synapses in the mammalian central nervous system. Gly concentrations at post-synaptic neurotransmitter receptors are regulated by Na+/Cl(-)-dependent Gly transporters, which are expressed in neurons and in glial cells. Recent evidence suggests that these transporters are promising targets for the treatment of psychiatric and neurological disorders, such as schizophrenia and pain. Here, recent research on the structure, regulation and pharmacology of mammalian Gly transporters is reviewed.     

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.     

The glutamatergic system outside the CNS and in cancer biology

Glutamate is a major excitatory neurotransmitter in the CNS. The signalling machinery consists of: glutamate receptors, which are responsible for signal input; plasma glutamate transporters, which are responsible for signal termination; and vesicular glutamate transporters for signal output through exocytic release. Recently, data have suggested that the glutamatergic system plays an important role in non-neuronal tissues. In addition, the expression of glutamatergic system has been implicated in tumour biology. This review outlines the evidence, which suggests that the glutamatergic system may have an important role in cancer biology.     

The glutamatergic system outside the CNS and in cancer biology

Glutamate is a major excitatory neurotransmitter in the CNS. The signalling machinery consists of: glutamate receptors, which are responsible for signal input; plasma glutamate transporters, which are responsible for signal termination; and vesicular glutamate transporters for signal output through exocytic release. Recently, data have suggested that the glutamatergic system plays an important role in non-neuronal tissues. In addition, the expression of glutamatergic system has been implicated in tumour biology. This review outlines the evidence, which suggests that the glutamatergic system may have an important role in cancer biology.