The Experts below are selected from a list of 10383 Experts worldwide ranked by ideXlab platform
Susan G Amara - One of the best experts on this subject based on the ideXlab platform.
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cysteine transport through Excitatory Amino Acid transporter 3 eaat3
PLOS ONE, 2014Co-Authors: Spencer D Watts, Christopher B Divito, Delany Torressalazar, Susan G AmaraAbstract:Excitatory Amino Acid transporters (EAATs) limit glutamatergic signaling and maintain extracellular glutamate concentrations below neurotoxic levels. Of the five known EAAT isoforms (EAATs 1-5), only the neuronal isoform, EAAT3 (EAAC1), can efficiently transport the uncharged Amino Acid L-cysteine. EAAT3-mediated cysteine transport has been proposed to be a primary mechanism used by neurons to obtain cysteine for the synthesis of glutathione, a key molecule in preventing oxidative stress and neuronal toxicity. The molecular mechanisms underlying the selective transport of cysteine by EAAT3 have not been elucidated. Here we propose that the transport of cysteine through EAAT3 requires formation of the thiolate form of cysteine in the binding site. Using Xenopus oocytes and HEK293 cells expressing EAAT2 and EAAT3, we assessed the transport kinetics of different substrates and measured transporter-associated currents electrophysiologically. Our results show that L-selenocysteine, a cysteine analog that forms a negatively-charged selenolate ion at physiological pH, is efficiently transported by EAATs 1-3 and has a much higher apparent affinity for transport when compared to cysteine. Using a membrane tethered GFP variant to monitor intracellular pH changes associated with transport activity, we observed that transport of either L-glutamate or L-selenocysteine by EAAT3 decreased intracellular pH, whereas transport of cysteine resulted in cytoplasmic alkalinization. No change in pH was observed when cysteine was applied to cells expressing EAAT2, which displays negligible transport of cysteine. Under conditions that favor release of intracellular substrates through EAAT3 we observed release of labeled intracellular glutamate but did not detect cysteine release. Our results support a model whereby cysteine transport through EAAT3 is facilitated through cysteine de-protonation and that once inside, the thiolate is rapidly re-protonated. Moreover, these findings suggest that cysteine transport is predominantly unidirectional and that reverse transport does not contribute to depletion of intracellular cysteine pools.
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Excitatory Amino Acid transporters: keeping up with glutamate.
Neurochemistry international, 2002Co-Authors: Susan G Amara, Andréia C. K. FontanaAbstract:Excitatory Amino Acid transporters (EAATs) are the primary regulators of extracellular glutamate concentrations in the central nervous system. Among the five known human EAAT subtypes, the glial carriers, EAAT1 and EAAT2 have the greatest impact on clearance of glutamate released during neurotransmission. Studies of carriers expressed on neurons, Purkinje cells and photoreceptor cells (EAAT3, EAAT4 and EAAT5, respectively) suggest more subtle roles for these subtypes in regulating excitability and signalling. The data suggest that EAA transporters may influence glutamatergic transmission by regulating the amount of glutamate available to activate pre- and post-synaptic metabotropic receptors and by altering neuronal excitability through a transporter-associated anion conductance that is activated by carrier substrates. Recent studies on structural, mechanistic and physiological aspects of carrier function in a variety of model systems and organisms have led to surprising insights into how Excitatory Amino Acid transporters shape cellular communication in the nervous system.
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a model for the topology of Excitatory Amino Acid transporters determined by the extracellular accessibility of substituted cysteines
Neuron, 2000Co-Authors: Rebecca P. Seal, Barbara H Leighton, Susan G AmaraAbstract:Excitatory Amino Acid transporters (EAATs) function as both substrate transporters and ligand-gated anion channels. Characterization of the transporter's general topology is the first requisite step in defining the structural bases for these distinct activities. While the first six hydrophobic domains can be readily modeled as conventional transmembrane segments, the organization of the C-terminal hydrophobic domains, which have been implicated in both substrate and ion interactions, has been controversial. Here, we report the results of a comprehensive evaluation of the C-terminal topology of EAAT1 determined by the chemical modification of introduced cysteine residues. Our data support a model in which two membrane-spanning domains flank a central region that is highly accessible to the extracellular milieu and contains at least one reentrant loop domain.
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Excitatory Amino Acid transporters: a family in flux.
Annual review of pharmacology and toxicology, 1999Co-Authors: Rebecca P. Seal, Susan G AmaraAbstract:As the most predominant Excitatory neurotransmitter, glutamate has the potential to influence the function of most neuronal circuits in the central nervous system. To limit receptor activation during signaling and prevent the overstimulation of glutamate receptors that can trigger excitotoxic mechanisms and cell death, extracellular concentrations of Excitatory Amino Acids are tightly controlled by transport systems on both neurons and glial cells. L-Glutamate is a potent neurotoxin, and the inadequate clearance of Excitatory Amino Acids may contribute to the neurodegeneration seen in a variety of conditions, including epilepsy, ischemia, and amyotrophic lateral sclerosis. To establish the contributions of carrier systems to the etiology of neurological disorders, and to consider their potential utility as therapeutic targets, a detailed understanding of transporter function and pharmacology is required. This review summarizes current knowledge of the structural and functional diversity of Excitatory Amino Acid transporters and explores how they might serve as targets for drug design.
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Excitatory Amino Acid transporters of the salamander retina identification localization and function
The Journal of Neuroscience, 1998Co-Authors: Scott Eliasof, Michael P Kavanaugh, Susan G Amara, Barbara H Leighton, Jeffrey L ArrizaAbstract:The rapid re-uptake of extracellular glutamate mediated by a family of high-affinity glutamate transporter proteins is essential to continued glutamatergic signaling and neuronal viability, but the contributions of individual transporter subtypes toward cellular physiology are poorly understood. Because the physiology of glutamate transport in the salamander retina has been well described, we have examined the expression and function of glutamate transporter subtypes in this preparation. cDNAs encoding five distinct salamander Excitatory Amino Acid transporter (sEAAT) subtypes were isolated, and their molecular properties and distributions of expression were compared. We report evidence that at least four distinct sEAAT subtypes are expressed in glial (Muller) cells. In addition, four of the five transporter subtypes are localized in neurons throughout the retina. The brightest immunostaining was seen in the synaptic regions of the inner and outer plexiform layers and in the outer nuclear layer. Using electrophysiological measurements in the Xenopus oocyte expression system, we also examined the pharmacology and ionic dependence of the four expressing transporter subtypes that make it possible to distinguish, on the basis of functional behavior, among the various subtypes. Although no simple correlation between transporter subtype and retinal cell physiology can be made, the diverse population of sEAAT transporter subtypes with unique localization and functional properties indicates that glutamate transporters play a wide variety of roles in retinal function and are likely to underlie both the uptake of glutamate by Muller cells and the glutamate-elicited chloride conductance involved in signal transduction by photoreceptors and bipolar cells.
Michael P Kavanaugh - One of the best experts on this subject based on the ideXlab platform.
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modeling of Excitatory Amino Acid transporters and clearance of synaptic cleft on millisecond time scale
Mathematical Modelling of Natural Phenomena, 2019Co-Authors: Denis Shchepakin, Leonid V Kalachev, Michael P KavanaughAbstract:Excitatory Amino Acid Transporters (EAATs) operate over wide time scales in the brain. They maintain low ambient concentrations of the primary Excitatory Amino Acid neurotransmitter glutamate, but they also seem to play a significant role in clearing glutamate from the synaptic cleft in the millisecond time-scale process of chemical communication that occurs between neurons. The detailed kinetic mechanisms underlying glutamate uptake and clearance remain incompletely understood. In this work we used a combination of methods to model EAAT kinetics and gain insight into the impact of transport on glutamate dynamics in a general sense. We derive reliable estimates of the turnover rates of the three major EAAT subtypes expressed in the mammalian cerebral cortex. Previous studies have provided transporter kinetic estimates that vary over an order of magnitude. The values obtained in this study are consistent with estimates that suggest the unitary transporter rates are approximately 20-fold slower than the time course of glutamate in the synapse. A combined diffusion/transport model provides a possible mechanism for the apparent discrepancy.
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modeling of Excitatory Amino Acid transporters and clearance of synaptic cleft on millisecond time scale
Mathematical Modelling of Natural Phenomena, 2019Co-Authors: Denis Shchepakin, Leonid V Kalachev, Michael P KavanaughAbstract:Excitatory Amino Acid Transporters (EAATs) operate over wide time scales in the brain. They maintain low ambient concentrations of the primary Excitatory Amino Acid neurotransmitter glutamate, but they also seem to play a significant role in clearing glutamate from the synaptic cleft in the millisecond time-scale process of chemical communication that occurs between neurons. The detailed kinetic mechanisms underlying glutamate uptake and clearance remain incompletely understood. In this work we used a combination of methods to model EAAT kinetics and gain insight into the impact of transport on glutamate dynamics in a general sense. We derive reliable estimates of the turnover rates of the three major EAAT subtypes expressed in the mammalian cerebral cortex. Previous studies have provided transporter kinetic estimates that vary over an order of magnitude. The values obtained in this study are consistent with estimates that suggest the unitary transporter rates are approximately 20-fold slower than the time course of glutamate in the synapse. A combined diffusion/transport model provides a possible mechanism for the apparent discrepancy.
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Excitatory Amino Acid transporters of the salamander retina identification localization and function
The Journal of Neuroscience, 1998Co-Authors: Scott Eliasof, Michael P Kavanaugh, Susan G Amara, Barbara H Leighton, Jeffrey L ArrizaAbstract:The rapid re-uptake of extracellular glutamate mediated by a family of high-affinity glutamate transporter proteins is essential to continued glutamatergic signaling and neuronal viability, but the contributions of individual transporter subtypes toward cellular physiology are poorly understood. Because the physiology of glutamate transport in the salamander retina has been well described, we have examined the expression and function of glutamate transporter subtypes in this preparation. cDNAs encoding five distinct salamander Excitatory Amino Acid transporter (sEAAT) subtypes were isolated, and their molecular properties and distributions of expression were compared. We report evidence that at least four distinct sEAAT subtypes are expressed in glial (Muller) cells. In addition, four of the five transporter subtypes are localized in neurons throughout the retina. The brightest immunostaining was seen in the synaptic regions of the inner and outer plexiform layers and in the outer nuclear layer. Using electrophysiological measurements in the Xenopus oocyte expression system, we also examined the pharmacology and ionic dependence of the four expressing transporter subtypes that make it possible to distinguish, on the basis of functional behavior, among the various subtypes. Although no simple correlation between transporter subtype and retinal cell physiology can be made, the diverse population of sEAAT transporter subtypes with unique localization and functional properties indicates that glutamate transporters play a wide variety of roles in retinal function and are likely to underlie both the uptake of glutamate by Muller cells and the glutamate-elicited chloride conductance involved in signal transduction by photoreceptors and bipolar cells.
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Excitatory Amino Acid transporter 5 a retinal glutamate transporter coupled to a chloride conductance
Proceedings of the National Academy of Sciences of the United States of America, 1997Co-Authors: Jeffrey L Arriza, Michael P Kavanaugh, Scott Eliasof, Susan G AmaraAbstract:Although a glutamate-gated chloride conductance with the properties of a sodium-dependent glutamate transporter has been described in vertebrate retinal photoreceptors and bipolar cells, the molecular species underlying this conductance has not yet been identified. We now report the cloning and functional characterization of a human Excitatory Amino Acid transporter, EAAT5, expressed primarily in retina. Although EAAT5 shares the structural homologies of the EAAT gene family, one novel feature of the EAAT5 sequence is a carboxy-terminal motif identified previously in N-methyl-d-aspartate receptors and potassium channels and shown to confer interactions with a family of synaptic proteins that promote ion channel clustering. Functional properties of EAAT5 were examined in the Xenopus oocyte expression system by measuring radiolabeled glutamate flux and two-electrode voltage clamp recording. EAAT5-mediated l-glutamate uptake is sodium- and voltage-dependent and chloride-independent. Transporter currents elicited by glutamate are also sodium- and voltage-dependent, but ion substitution experiments suggest that this current is largely carried by chloride ions. These properties of EAAT5 are similar to the glutamate-elicited chloride conductances previously described in retinal neurons, suggesting that the EAAT5-associated chloride conductance may participate in visual processing.
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ion fluxes associated with Excitatory Amino Acid transport
Neuron, 1995Co-Authors: Jacques I Wadiche, Susan G Amara, Michael P KavanaughAbstract:Flux of substrate and charge mediated by three cloned Excitatory Amino Acid transporters widely expressed in human brain were studied in voltage-clamped Xenopus oocytes. Superfusion of L-glutamate or D-aspartate resulted in currents due in part to electrogenic Na+ cotransport, which contributed 1 net positive charge per transport cycle. A significant additional component of the currents was due to activation of a reversible anion flux that was not thermodynamically coupled to Amino Acid transport. The selectivity sequence of this ligand-activated conductance was NO3- > 1- > Br- > Cl- > F-. The results suggest that these proteins mediate both transporter- and channel-like modes of permeation, providing a potential mechanism for dampening cell excitability, in addition to removal of transmitter.
Glenn M Toney - One of the best experts on this subject based on the ideXlab platform.
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sympathoexcitation by pvn injected bicuculline requires activation of Excitatory Amino Acid receptors
Hypertension, 2003Co-Authors: Qing Hui Chen, Joseph R Haywood, Glenn M ToneyAbstract:Acute blockade of γ-Aminobutyric Acid (GABA)-A receptors in the hypothalamic paraventricular nucleus (PVN) increases mean arterial pressure (MAP), heart rate (HR), and sympathetic nerve activity (SNA). However, the underlying neural mechanisms have not been fully determined. We tested the hypothesis that responses to GABA-A receptor blockade in the PVN require activation of local ionotropic Excitatory Amino Acid (EAA) receptors. MAP, HR, and renal SNA responses to unilateral PVN microinjection of bicuculline methobromide (BIC, 0.1 nmol) were recorded before and after ipsilateral PVN injection of either vehicle (saline), the nonselective ionotropic EAA receptor antagonist kynurenate (KYN), the NMDA receptor antagonist D(-)-2-Amino-5-phosphonopentanoic Acid (AP5), or the non-NMDA receptor antagonist 2,3-dioxo-6-nitro-1,2,3,4-tetrahydrobenzo[f]quinoxaline-7-sulfonamide disodium (NBQX). Responses to PVN-injected BIC were unaltered by vehicle injection. In contrast, injection of KYN (7.2 nmol; n=4) nearly abolished ABP and renal SNA responses to BIC ( P P P P
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sympathoexcitation by pvn injected bicuculline requires activation of Excitatory Amino Acid receptors
Hypertension, 2003Co-Authors: Qing Hui Chen, Joseph R Haywood, Glenn M ToneyAbstract:Acute blockade of gamma-Aminobutyric Acid (GABA)-A receptors in the hypothalamic paraventricular nucleus (PVN) increases mean arterial pressure (MAP), heart rate (HR), and sympathetic nerve activity (SNA). However, the underlying neural mechanisms have not been fully determined. We tested the hypothesis that responses to GABA-A receptor blockade in the PVN require activation of local ionotropic Excitatory Amino Acid (EAA) receptors. MAP, HR, and renal SNA responses to unilateral PVN microinjection of bicuculline methobromide (BIC, 0.1 nmol) were recorded before and after ipsilateral PVN injection of either vehicle (saline), the nonselective ionotropic EAA receptor antagonist kynurenate (KYN), the NMDA receptor antagonist D(-)-2-Amino-5-phosphonopentanoic Acid (AP5), or the non-NMDA receptor antagonist 2,3-dioxo-6-nitro-1,2,3,4-tetrahydrobenzo[f]quinoxaline-7-sulfonamide disodium (NBQX). Responses to PVN-injected BIC were unaltered by vehicle injection. In contrast, injection of KYN (7.2 nmol; n=4) nearly abolished ABP and renal SNA responses to BIC (P<0.01) and significantly attenuated (P<0.05) HR responses as well. Similarly, graded doses of AP5 (0.6, 3, and 6 nmol) and NBQX (0.26, 1.3, and 2.6 nmol) reduced responses to PVN-injected BIC in a dose-related manner, with the 3 nmol (n=7) and 1.3 nmol (n=6) doses producing maximal effects (P<0.05). KYN, AP5, and NBQX did not affect baseline parameters. Effects of a cocktail containing AP5 (3 nmol) and NBQX (1.3 nmol) were greater (P<0.01) than either antagonist alone and were not statistically different from KYN. These data indicate that cardiovascular and renal sympathetic responses to acute GABA-A receptor blockade in the PVN require local actions of EAAs at both NMDA and non-NMDA receptors.
Maria J Vidal - One of the best experts on this subject based on the ideXlab platform.
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l arginine potentiates Excitatory Amino Acid induced seizures elicited in the deep prepiriform cortex
European Journal of Pharmacology, 1993Co-Authors: Giovanbattista De Sarro, Eugenio Donato Di Paola, Angela De Sarro, Maria J VidalAbstract:Microinjection of N-methyl-D-aspartate (NMDA; 1 and 2.5 nmol) or kainate (KA; 50 pmol) into the deep prepiriform cortex elicited behavioral signs of seizure activity. No epileptiform activity was observed after deep prepiriform cortex microinjection of either L-arginine (L-Arg, 5 and 10 nmol) or its D-enantiomer, D-arginine (D-Arg, 2.5-10 nmol). However, both the seizure score and the incidence of electroencephalographic (EEG) epileptic discharges elicited by NMDA (1 and 2.5 nmol) and KA (50 pmol) were significantly increased by L- but not D-Arg. The facilitatory effects of L-Arg on seizure activity elicited by both NMDA and KA were dose-dependent and could be prevented by co-administration of L-Arg (10 nmol) and the nitric oxide (NO) synthase inhibitor, N omega-nitro-L-arginine methyl ester (L-NAME, 20 nmol). Motor and electrocortical seizures were observed after microinjection of the NO donor sodium nitroprusside (SNP; 5 to 20 nmol) into the deep prepiriform cortex. Infusion of methylene blue (20 nmol), a soluble guanylate cyclase inhibitor, protected against SNP-induced seizures. Furthermore, prior infusion of a subconvulsant dose of SNP into the deep prepiriform cortex significantly potentiated the seizure activity elicited by either NMDA (1 and 2.5 nmol) or KA (50 pmol). These results support the proposal that NO is formed from L-Arg upon Excitatory Amino Acid receptor activation within the deep prepiriform cortex, thereby contributing to the genesis of seizure activity.
S M Roychowdhury - One of the best experts on this subject based on the ideXlab platform.
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reflex related activation of putative pain facilitating neurons in rostral ventromedial medulla requires Excitatory Amino Acid transmission
Neuroscience, 1997Co-Authors: Mary M Heinricher, S M RoychowdhuryAbstract:Abstract Although the importance of the rostral ventromedial medulla in pain modulation is generally accepted, the recognition that it can exert both pain facilitating and pain inhibiting influences, and that its constituent neuronal population is physiologically and pharmacologically heterogeneous, is relatively recent. A class of neuron which may be a source of facilitating influences from the rostral ventromedial medulla has been identified in electrophysiological experiments. These neurons, termed “on-cells,” are characterized by a sudden burst of activity beginning just before nocifensive reflexes. This burst of firing is thought to be a significant factor in brainstem control of nociceptive transmission under physiological conditions. The aim of the present study was to determine whether an Excitatory Amino Acid is involved in generation of the reflex-related burst that defines on-cells, and more generally, to examine the role of Excitatory Amino Acid neurotransmitters within the rostral ventromedial medulla of the rat. Iontophoretic application of the broad-spectrum Excitatory Amino Acid receptor antagonist kynurenate significantly reduced the reflex-related on-cell burst, whereas ongoing firing was unaffected. Spontaneous activity of other medullary neurons was unchanged. These data demonstrate that release of an endogenous Excitatory Amino Acid neurotransmitter is necessary for the activation of on-cells that is associated with nocifensive reflexes. In contrast, these receptors evidently play a much less significant role in maintaining the ongoing activity of any cell class in the rostral ventromedial medulla in lightly anaesthetized rats.
