Glycine transporters and synaptic function

Glycine is an inhibitory neurotransmitter that is mainly active in the caudal areas of the CNS. However, glycine also participates in excitatory neurotransmission since it is a co-agonist of the NMDA subtype of glutamate receptors. The concentration of glycine at synapses is mainly controlled by two sodium and chloride dependent transporters, GLYT1 and GLYT2, proteins that display a complementary distribution and activity in the nervous system. Our understanding of the physiological role of these transporters has advanced recently, thanks to the development of specific inhibitors and the generation of mice defective in the corresponding genes. In addition, the three-dimensional resolution of the structure of a bacterial homologue has shed light on the mechanisms of glycine transport. It is likely that this knowledge will prove to be useful for the development of drugs with antipsychotic, procognitive or analgesic properties.     

On cotransmission & neurotransmitter phenotype plasticity

Most neurons in the nervous system appear to contain and release more than one chemical acting as a neurotransmitter or neuromodulator. Cotransmission can therefore be considered the rule rather than the exception. Indeed, cotransmission of a classical neurotransmitter and a peptide is a ubiquitous phenomenon, but several neuron types can also contain more than one classical neurotransmitter [glutamate, gamma-amino butyric acid (GABA), acetylcholine, dopamine, etc.]. Although the expression of peptide cotransmitters is known to be highly regulated in response to various physiological, chemical and pathological signals, new data now suggest that a similar situation prevails in neurons that co-release two classical transmitters. In this review we will consider a number of recently described examples of cotransmission implicating more than one classical neurotransmitter. We will also consider new data showing that during development and later in adulthood, as well as in the context of disease, the neurotransmitter phenotype of neurons can be highly plastic as revealed by changes in the expression of neurotransmitter synthesis enzymes and vesicular neurotransmitter transporters.     

The role of glutamate transporters in neurodegenerative diseases and potential opportunities for intervention

Extracellular concentrations of the predominant excitatory neurotransmitter, glutamate, and related excitatory amino acids are maintained at relatively low levels to ensure an appropriate signal-to-noise ratio and to prevent excessive activation of glutamate receptors that can result in cell death. The latter phenomenon is known as 'excitotoxicity' and has been associated with a wide range of acute and chronic neurodegenerative disorders, as well as disorders that result in the loss of non-neural cells such as oligodendroglia in multiple sclerosis. Unfortunately clinical trials with glutamate receptor antagonists that would logically seem to prevent the effects of excessive receptor activation have been associated with untoward side effects or little clinical benefit. In the mammalian CNS, the extracellular concentrations of glutamate are controlled by two types of transporters; these include a family of Na(+)-dependent transporters and a cystine-glutamate exchange process, referred to as system X(c)(-). In this review, we will focus primarily on the Na(+)-dependent transporters. A brief introduction to glutamate as a neurotransmitter will be followed by an overview of the properties of these transporters, including a summary of the presumed physiologic mechanisms that regulate these transporters. Many studies have provided compelling evidence that impairing the function of these transporters can increase the sensitivity of tissue to deleterious effects of aberrant activation of glutamate receptors. Over the last decade, it has become clear that many neurodegenerative disorders are associated with a change in localization and/or expression of some of the subtypes of these transporters. This would suggest that therapies directed toward enhancing transporter expression might be beneficial. However, there is also evidence that glutamate transporters might increase the susceptibility of tissue to the consequences of insults that result in a collapse of the electrochemical gradients required for normal function such as stroke. In spite of the potential adverse effects of upregulation of glutamate transporters, there is recent evidence that upregulation of one of the glutamate transporters, GLT-1 (also called EAAT2), with beta-lactam antibiotics attenuates the damage observed in models of both acute and chronic neurodegenerative disorders. While it seems somewhat unlikely that antibiotics specifically target GLT-1 expression, these studies identify a potential strategy to limit excitotoxicity. If successful, this type of approach could have widespread utility given the large number of neurodegenerative diseases associated with decreases in transporter expression and excitotoxicity. However, given the massive effort directed at developing glutamate receptor agents during the 1990s and the relatively modest advances to date, one wonders if we will maintain the patience needed to carefully understand the glutamatergic system so that it will be successfully targeted in the future.     

Glutamate forward and reverse transport: from molecular mechanism to transporter-mediated release after ischemia

Glutamate transporters remove the excitatory neurotransmitter glutamate from the extracellular space after neurotransmission is complete, by taking glutamate up into neurons and glia cells. As thermodynamic machines, these transporters can also run in reverse, releasing glutamate into the extracellular space. Because glutamate is excitotoxic, this transporter-mediated release is detrimental to the health of neurons and axons, and it, thus, contributes to the brain damage that typically follows a stroke. This review highlights current ideas about the molecular mechanisms underlying glutamate uptake and glutamate reverse transport. It also discusses the implications of transporter-mediated glutamate release for cellular function under physiological and patho-physiological conditions.     

Glutamate Transporters and Mitochondria: Signaling,Co-compartmentalization,Functional Coupling,and Future Directions

In addition to being an amino acid that is incorporated into proteins, glutamate is the most abundant neurotransmitter in the mammalian CNS, the precursor for the inhibitory neurotransmitter γ-aminobutyric acid, and one metabolic step from the tricarboxylic acid cycle intermediate α-ketoglutarate. Extracellular glutamate is cleared by a family of Na⁺-dependent transporters. These transporters are variably expressed by all cell types in the nervous system, but the bulk of clearance is into astrocytes. GLT-1 and GLAST (also called EAAT2 and EAAT1) mediate this activity and are extremely abundant proteins with their expression enriched in fine astrocyte processes. In this review, we will focus on three topics related to these astrocytic glutamate transporters. First, these transporters co-transport three Na⁺ ions and a H⁺ with each molecule of glutamate and counter-transport one K⁺; they are also coupled to a Cl^? conductance. The movement of Na⁺ is sufficient to cause profound astrocytic depolarization, and the movement of H⁺ is linked to astrocytic acidification. In addition, the movement of Na⁺ can trigger the activation of Na⁺ co-transporters (e.g. Na⁺–Ca²⁺ exchangers). We will describe the ways in which these ionic movements have been linked as signals to brain function and/or metabolism. Second, these transporters co-compartmentalize with mitochondria, potentially providing a mechanism to supply glutamate to mitochondria as a source of fuel for the brain. We will provide an overview of the proteins involved, discuss the evidence that glutamate is oxidized, and then highlight some of the un-resolved issues related to glutamate oxidation. Finally, we will review evidence that ischemic insults (stroke or oxygen/glucose deprivation) cause changes in these astrocytic mitochondria and discuss the ways in which these changes have been linked to glutamate transport, glutamate transport-dependent signaling, and altered glutamate metabolism. We conclude with a broader summary of some of the unresolved issues.

     

How did the neurotransmitter cross the bilayer? A closer view

Plasma membrane neurotransmitter transporters for monoamines, GABA, glycine and excitatory amino acids are homologous to two sizable families of bacterial amino acid transporters. Recently, a high resolution structure was determined for a thermophilic glutamate transporter. Also, a bacterial tryptophan transporter related to the family of biogenic amine neurotransmitter transporters was functionally expressed. Structural insights from these and other bacterial transporters will help to rationalize the mechanisms for the increasingly complex functions that have been described for mammalian transporters, in addition to their modes of regulation. We touch on recent insights into the functions of neurotransmitter transporters in their physiological contexts.     

Glutamate Transporters: Expression and Function in Oligodendrocytes

Glutamate, the main excitatory neurotransmitter of the vertebrate central nervous system (CNS), is well known as a regulator of neuronal plasticity and neurodevelopment. Such glutamate function is thought to be mediated primarily by signaling through glutamate receptors. Thus, it requires a tight regulation of extracellular glutamate levels and a fine-tuned homeostasis that, when dysregulated, has been associated with a wide range of central pathologies including neuropsychiatric, neurodevelopmental, and neurodegenerative disorders. In the mammalian CNS, extracellular glutamate levels are controlled by a family of sodium-dependent glutamate transporters belonging to the solute carrier family 1 (SLC1) that are also referred to as excitatory amino acid transporters (EAATs). The presumed main function of EAATs has been best described in the context of synaptic transmission where EAATs expressed by astrocytes and neurons effectively regulate extracellular glutamate levels so that synapses can function independently. There is, however, increasing evidence that EAATs are expressed by cells other than astrocytes and neurons, and that they exhibit functions beyond glutamate clearance. In this review, we will focus on the expression and functions of EAATs in the myelinating cells of the CNS, oligodendrocytes. More specifically, we will discuss potential roles of oligodendrocyte-expressed EAATs in contributing to extracellular glutamate homeostasis, and in regulating oligodendrocyte maturation and CNS myelination by exerting signaling functions that have traditionally been associated with glutamate receptors. In addition, we will provide some examples for how dysregulation of oligodendrocyte-expressed EAATs may be involved in the pathophysiology of neurologic diseases.

     

Glial glutamate transporters: New actors in brain signaling

Glutamate, the main excitatory amino acid in the vertebrate brain, is critically involved in most of the physiological functions of the central nervous system. It has traditionally been assumed that glutamate triggers a wide array of signaling cascades through the activation of specific membrane receptors. The extracellular levels are tightly regulated to prevent neurotoxic insults. Electrogenic Na(+)-dependent glial glutamate transporters remove the bulk of the neurotransmitter from the synaptic cleft. An exquisitely ordered coupling between glutamatergic neurons and surrounding glia cells is fundamental for excitatory transmission. The glutamate/glutamine and astrocyte/neuron lactate shuttles provide the biochemical framework of this compulsory association. In this context, recent advances show that glial glutamate transporters act as signal transducers that regulate the expression of proteins involved in their compartmentalization with neurons in the so-called tripartite synapse.     

多巴胺能神经系统对睡眠-觉醒和认知的调控作用

人的精神活动高级而又复杂,至今仍是未解之谜。目前研究认为多巴胺作为脑内重要神经递质,参与调节人的精神活动和运动功能,尤其在睡眠的主动性神经调节过程,以及学习记忆等认知功能的神经环路中,多巴胺都发挥着不可替代的作用。本文将通过对多巴胺神经系统,睡眠,认知功能的概述,以及通过对多巴胺神经系统与睡眠-觉醒系统和认知功能的解剖学联系的简述,结合多巴胺神经元、多巴胺受体及多巴胺转运体等不同角度分别阐述其对睡眠-觉醒和认知功能的调控作用,以期揭开人类精神活动的产生机制的一层面纱,以及对多巴胺药物对神经退行性变疾病的治疗靶点提供一定的理论支持。     

Neuronal-induced and glutamate-dependent activation of glial glutamate transporter function

The activity of high-affinity glutamate transporters is essential for the normal function of the mammalian central nervous system. Using a combined pharmacological, confocal immunocytochemical, enzyme-based microsensor and fluorescence imaging approach, we examined glutamate uptake and transporter protein localization in single astrocytes of neuron-containing and neuron-free microislands prior to pre-synaptic transmitter secretion and during functional neuronal activity. Here, we report that the presence or absence of neurons strikingly affects the uptake capacity of the astroglial glutamate transporters GLT1 and GLAST1. Induction of transporter function is activated by neurons and this effect is mimicked by pre-incubation of astrocytes with micromolar concentrations of glutamate. Moreover, increased glutamate transporter activation is reproduced by endogenous release of glutamate via activation of neuronal nicotinic receptors. The increase in transport activity is dependent on neuronal release of glutamate, is associated with the local redistribution (clustering) of GLT1 and GLAST1 but is independent of transporter synthesis and of glutamate receptor activation. Together, these results suggest an activity-dependent neuronal feedback system for rapid astroglial glutamate transporter regulation where neuron-derived glutamate is the physiological signal that triggers transporter function.     

谷氨酸转运体与疼痛

中枢神经系统谷氨酸生理浓度主要依赖神经细胞和神经胶质细胞上谷氨酸转运体维持,谷氨酸转运体的功能紊乱会导致谷氨酸的累积。谷氨酸转运体在吗啡镇痛及耐受中扮演一定的角色,并在神经病理性痛中发挥重要作用。谷氨酸转运体可能作为治疗疼痛的一个潜在的药物靶点。     

Contribution of astrocytes to synaptic transmission in the rat supraoptic nucleus

Astrocytes, besides supporting metabolic and scaffolding functions, play a prominent role in the modulation of neuronal communication. In particular, they are responsible for clearing synaptically-released glutamate via highly specific transporters located on their plasma membrane. Since glutamate is the main excitatory neurotransmitter in the central nervous system (CNS), astrocytes are likely to play a central role in the regulation of synaptic processing and overall cellular excitability. We recently investigated the influence of astrocytes on glutamatergic and GABAergic transmission in the rat supraoptic nucleus (SON) of the hypothalamus. This nucleus is part of the hypothalamus-neurohypophysial system (HNS), which constitutes a conspicuous example of activity-dependent neuroglial plasticity, in which certains physiological conditions, such as parturition, lactation, and dehydration are accompanied by a structural remodeling of the neurones, their synaptic inputs and their surrounding glia. The use of pharmacological inhibitors of glutamate transporters on this model, in which a physiological change in the astrocyte environment occurs, has brought new insights on the contribution of astrocytes to both excitatory and inhibitory neurotransmissions. The astrocytic environment of neurons appears to control glutamate uptake and diffusion in the extracellular space. This has direct repercussions on the tonic level of activation of presynaptic glutamate receptors and, as a consequence, on the release of neurotransmitter. This short review summarizes data obtained so far, which clearly support the view that astrocytes are indeed a third partner in synaptic transmission, and which show that the supraoptic nucleus represents a remarkable model to study dynamic physiological interactions between astrocytes and neurons.     

Physical and functional interaction of NCX1 and EAAC1 transporters leading to glutamate-enhanced ATP production in brain mitochondria

Glutamate is emerging as a major factor stimulating energy production in CNS. Brain mitochondria can utilize this neurotransmitter as respiratory substrate and specific transporters are required to mediate the glutamate entry into the mitochondrial matrix. Glutamate transporters of the Excitatory Amino Acid Transporters (EAATs) family have been previously well characterized on the cell surface of neuronal and glial cells, representing the primary players for glutamate uptake in mammalian brain. Here, by using western blot, confocal microscopy and immunoelectron microscopy, we report for the first time that the Excitatory Amino Acid Carrier 1 (EAAC1), an EAATs member, is expressed in neuronal and glial mitochondria where it participates in glutamate-stimulated ATP production, evaluated by a luciferase-luciferin system. Mitochondrial metabolic response is counteracted when different EAATs pharmacological blockers or selective EAAC1 antisense oligonucleotides were used. Since EAATs are Na(+)-dependent proteins, this raised the possibility that other transporters regulating ion gradients across mitochondrial membrane were required for glutamate response. We describe colocalization, mutual activity dependency, physical interaction between EAAC1 and the sodium/calcium exchanger 1 (NCX1) both in neuronal and glial mitochondria, and that NCX1 is an essential modulator of this glutamate transporter. Only NCX1 activity is crucial for such glutamate-stimulated ATP synthesis, as demonstrated by pharmacological blockade and selective knock-down with antisense oligonucleotides. The EAAC1/NCX1-dependent mitochondrial response to glutamate may be a general and alternative mechanism whereby this neurotransmitter sustains ATP production, since we have documented such metabolic response also in mitochondria isolated from heart. The data reported here disclose a new physiological role for mitochondrial NCX1 as the key player in glutamate-induced energy production.     

Glutamate uptake in synaptic plasticity: from mollusc to mammal

A great deal of research has been directed toward understanding the cellular mechanisms underlying synaptic plasticity and memory formation. To this point, most research has focused on the more "active" components of synaptic transmission: presynaptic transmitter release and postsynaptic transmitter receptors. Little work has been done characterizing the role neurotransmitter transporters might play during changes in synaptic efficacy. We review several new experiments that demonstrate glutamate transporters are regulated during changes in the efficacy of glutamatergic synapses. This regulation occurred during long-term facilitation of the sensorimotor synapse of Aplysia and long-term potentiation of the Schaffer-collateral synapse of the rat. We propose that glutamate transporters are "co-regulated" with other molecules/processes involved in synaptic plasticity, and that this process is phylogenetically conserved. These new findings indicate that glutamate transporters most likely play a more active role in neurotransmission than previously believed.     

The chloride permeation pathway of a glutamate transporter and its proximity to the glutamate translocation pathway

Excitatory amino acid transporters (EAATs) regulate glutamate concentrations in the brain to maintain normal excitatory synaptic transmission. A widely accepted view of transporters is that they consist of a pore with alternating access to the intracellular and extracellular solutions, which serves to couple ion movement to the movement of substrate. However, recent observations that EAATs, and also a number of other neurotransmitter transporters, can also function as ligand-gated chloride channels have blurred the distinctions between transporters and ion channels. Here we show that mutations in the second transmembrane domain (TM2) of EAAT1 alter anion permeation properties without affecting glutamate transport and that a number of TM2 residues are accessible to the external aqueous solution. Furthermore, we demonstrate that the extracellular edge of TM2 is in close proximity to a membrane-associated domain that influences glutamate transport. This study will provide the foundation for beginning to understand how transporters can function as both transporters and ion channels.     

Aspartate-444 is essential for productive substrate interactions in a neuronal glutamate transporter

In the central nervous system, electrogenic sodium- and potassium-coupled glutamate transporters terminate the synaptic actions of this neurotransmitter. In contrast to acidic amino acids, dicarboxylic acids are not recognized by glutamate transporters, but the related bacterial DctA transporters are capable of transporting succinate and other dicarboxylic acids. Transmembrane domain 8 contains several residues that differ between these two types of transporters. One of these, aspartate-444 of the neuronal glutamate transporter EAAC1, is conserved in glutamate transporters, but a serine residue occupies this position in DctA transporters. When aspartate-444 is mutated to serine, cysteine, alanine, or even to glutamate, uptake of D-[(3)H]-aspartate as well as the inwardly rectifying steady-state currents induced by acidic amino acids is impaired. Even though succinate was not capable of inducing any steady-state transport currents, the dicarboxylic acid inhibited the sodium-dependent transient currents by the mutants with a neutral substitution at position 444. In the neutral substitution mutants inhibition of the transients was also observed with acidic amino acids. In the D444E mutant, acidic amino acids were potent inhibitors of the transient currents, whereas the apparent affinity for succinate was lower by at least three orders of magnitude. Even though L-aspartate could bind to D444E with a high apparent affinity, this binding resulted in inhibition rather than stimulation of the uncoupled anion conductance. Thus, a carboxylic acid-containing side chain at position 444 prevents the interaction of glutamate transporters with succinate, and the presence of aspartate itself at this position is crucial for productive substrate binding compatible with substrate translocation.     

Synaptic vesicles: key organelles involved in neurotransmission

1. This article summarizes some of the recent advances in the understanding of structural and functional properties of isolated small synaptic vesicles (SSV) from mammalian brain. 2. SSV contain a set of integral membrane proteins which are highly specific for this organelle and which occur on all SSV of the central and peripheral nervous system irrespective of their transmitter content. In contrast, these proteins are absent from the membrane of peptide-containing large dense-core vesicles indicating that the two types of organelle have a different membrane composition. The availability of antibodies for these proteins has allowed the evaluation of the purity of vesicle preparations which is instrumental for functional studies. 3. Recent advances in the study of neurotransmitter uptake have revealed that SSV contain specific carrier systems for glutamate and GABA. They are different from the transporters of the plasma membrane, and are dependent on the energy of a proton electrochemical gradient. The uptake of glutamate has been characterized in some detail and the mechanistic and physiological implications of these findings are discussed.     

Glycine neurotransmitter transporters: an update

Glycine accomplishes several functions as a transmitter in the central nervous system (CNS). As an inhibitory neurotransmitter, it participates in the processing of motor and sensory information that permits movement, vision, and audition. This action of glycine is mediated by the strychnine-sensitive glycine receptor, whose activation produces inhibitory post-synaptic potentials. In some areas of the CNS, glycine seems to be co-released with GABA, the main inhibitory amino acid neurotransmitter. In addition, glycine modulates excitatory neurotransmission by potentiating the action of glutamate at N-methyl-D-aspartate (NMDA) receptors. It is believed that the termination of the different synaptic actions of glycine is produced by rapid re-uptake through two sodium-and-chloride-coupled transporters, GLYT1 and GLYT2, located in the plasma membrane of glial cells or pre-synaptic terminals, respectively. Glycine transporters may become major targets for therapeutic of pathological alterations in synaptic function. This article reviews recent progress on the study of the molecular heterogeneity, localization, function, structure, regulation and pharmacology of the glycine transporter proteins.     

Glycine neurotransmitter transporters: an update

Glycine accomplishes several functions as a transmitter in the central nervous system(CNS). As an inhibitory neurotransmitter, it participates in the processing of motor and sensory information that permits movement, vision, and audition. This action of glycine is mediated by the strychnine-sensitive glycine receptor, whose activation produces inhibitory post-synaptic potentials. In some areas of the CNS, glycine seems to be co-released with GABA, the main inhibitory amino acid neurotransmitter. In addition, glycine modulates excitatory neurotransmission by potentiating the action of glutamate at N-methyl-D-aspartate (NMDA) receptors. It is believed that the termination of the different synaptic actions of glycine is produced by rapid reuptake through two sodium-and-chloride-coupled transporters, GLYT1 and GLYT2, located in the plasma membrane of glial cells or pre-synaptic terminals, respectively. Glycine transporters may become major targets for therapeutic of pathological alterations in synaptic function. This article reviews recent progress on the study of the molecular heterogeneity, localization, function, structure, regulation and pharmacology of the glycine transporter     

The uncoupled chloride conductance of a bacterial glutamate transporter homolog

Glutamate transporters (EAATs) are pivotal in mammalian synaptic transmission, tightly regulating synaptic levels of this excitatory neurotransmitter. In addition to coupled glutamate transport, the EAATs also show an uncoupled Cl(-) conductance, whose physiological importance has recently been demonstrated. Little is yet known about the molecular mechanism of chloride permeation. Here we show that Glt(Ph), a bacterial EAAT homolog whose structure has been determined, displays an uncoupled Cl(-) conductance that can determine the rate of substrate uptake. A mutation analogous to one known to specifically affect Cl(-) movement in EAAT1 has similar effects on Glt(Ph), suggesting that this protein is an excellent structural model for understanding Cl(-) permeation through the EAATs. We also observed an uncoupled Cl(-) conductance in another bacterial EAAT homolog but not in a homolog of the Na(+)/Cl(-)-coupled neurotransmitter transporters.