scholarly journals The Conformationally Sensitive Spatial Distance Between the TM3-4 Loop and Transmembrane Segment 7 in the Glutamate Transporter Revealed by Paired-Cysteine Mutagenesis

2021 ◽  
Vol 9 ◽  
Author(s):  
Qi Qu ◽  
Ji Wang ◽  
Guiping Li ◽  
Rongqing Chen ◽  
Shaogang Qu
Keyword(s):  
Amino Acid ◽  
Excitatory Amino Acid ◽  
Glutamate Transporter ◽  
Synaptic Cleft ◽  
Spatial Distance ◽  
Cross Linking ◽  
Amino Acid Transporters ◽  
Transport Activity ◽  
Transmembrane Segment ◽  
Excitatory Amino Acid Transporter

Excitatory amino acid transporters can maintain extracellular glutamate concentrations lower than neurotoxic levels by transferring neurotransmitters from the synaptic cleft into surrounding glial cells and neurons. Previous work regarding the structural studies of GltPh, GltTK, excitatory amino acid transporter 1 (EAAT1), EAAT3 and alanine serine cysteine transporter 2 described the transport mechanism of the glutamate transporter in depth. However, much remains unknown about the role of the loop between transmembrane segment 3 and 4 during transport. To probe the function of this loop in the transport cycle, we engineered a pair of cysteine residues between the TM3-TM4 loop and TM7 in cysteine-less EAAT2. Here, we show that the oxidative cross-linking reagent CuPh inhibits transport activity of the paired mutant L149C/M414C, whereas DTT inhibits the effect of CuPh on transport activity of L149C/M414C. Additionally, we show that the effect of cross-linking in the mutant is due to the formation of the disulfide bond within the molecules of EAAT2. Further, L-glutamate or KCl protect, and D,L-threo-β-benzyloxy-aspartate (TBOA) increases, CuPh-induced inhibition in the L149C/M414 mutant, suggesting that the L149C and M414C cysteines are closer or farther away in the outward- or inward-facing conformations, respectively. Together, our findings provide evidence that the distance between TM3-TM4 loop and TM7 alter when substrates are transported.

Effects of the excitatory amino acid transporter subtype 2 (EAAT-2) inducer ceftriaxone on different pain modalities in rat

2011 ◽  
Vol 2 (3) ◽  
pp. 132-136 ◽  
Author(s):  
Laila Eljaja ◽  
Ole J. Bjerrum ◽  
Per Hartvig Honoré ◽  
Bjarke Abrahamsen
Keyword(s):  
Neuropathic Pain ◽  
Amino Acid ◽  
Inflammatory Responses ◽  
Excitatory Amino Acid ◽  
Synaptic Cleft ◽  
Spinal Nerve ◽  
Thermal Stimulation ◽  
Amino Acid Transporters ◽  
Excitatory Amino Acid Transporter ◽  
Chemical Structures

AbstractGlutamate is the major excitatory amino acid in the mammalian CNS and is involved in transmission of pain together with processes for cognition, memory and learning. In order to terminate glutamatergic neurotransmission and avoid excitotoxic damage, a balanced glutamate homeostasis is of critical importance. The level of glutamate in the synaptic cleft is regulated through the action of five subtypes of excitatory amino acid transporters (EAAT1-5). Ceftriaxone, a β-lactam, induces EAAT-2 and has proven effect for the treatment of neuropathic pain. This pilot study investigated the effects of ceftriaxone upon acute and inflammatory pain and additionally, the analgesic effect of ceftriaxone after introduction of neuropathic pain.MethodsRats were tested before, during and after treatment of ceftriaxone for changes in response to both mechanical and thermal stimuli, using calibrated von Frey filaments and Hargreaves instrument, respectively. Inflammatory responses were investigated by assessing the response to intra-plantar injections of formalin; lastly, neuropathic pain was introduced using the spinal nerve ligation (SNL) model after which changes in both mechanical and thermal responses were again investigated.ResultsA significant increase in mechanical withdrawal threshold was observed following acute pain inducement in ceftriaxone treated rats. A marked increase in thermal withdrawal latency was also observed. In response to intra plantar administered formalin, ceftriaxone delayed the intensity of nocifensive behaviours. Applying the SNL model of neuropathic pain on naive rats created significant mechanical allodynia, but only a negligibly different response to thermal stimulation. After treatment with ceftriaxone the treated rats developed a hypoalgesic response to thermal stimulation, whilst the response to mechanical pain was insignificant.ConclusionIn conclusion, ceftriaxone clearly interfered in the transmission of noxious signalling and proved in this study to have an effect upon acute thermal and mechanical pain thresholds as well as pathologic pain conditions. The present results are a piece in the large puzzle where administration route, dosage and pain models must be thoroughly investigated before a study can be planned for a proof of concept in different clinical pain states.ImplicationsThe current study demonstrates that ceftriaxone has a mitigating effect upon many pain modalities including acute and inflammatory, and that these modalities should be included in future studies characterising the anti-nociceptive effect of beta-lactams such as ceftriaxone. The fact that β-lactams also has antibiotic properties implies that similar chemical structures could be identified with the positive effect upon expression levels of EAAT2, but lacking the antibiotic side effect.

scholarly journals Recent Advance in the Relationship between Excitatory Amino Acid Transporters and Parkinson’s Disease

2016 ◽  
Vol 2016 ◽  
pp. 1-8 ◽  
Author(s):  
Yunlong Zhang ◽  
Feng Tan ◽  
Pingyi Xu ◽  
Shaogang Qu
Keyword(s):  
Parkinson’S Disease ◽  
Amino Acid ◽  
Animal Models ◽  
Substantia Nigra ◽  
Excitatory Amino Acid ◽  
Synaptic Cleft ◽  
Dopamine Neurons ◽  
Amino Acid Transporters ◽  
Excitatory Amino Acid Transporters ◽  
And Function

Parkinson’s disease (PD) is the most common movement disorder disease in the elderly and is characterized by degeneration of dopamine neurons and formation of Lewy bodies. Glutamate is the major excitatory neurotransmitter in the central nervous system (CNS). If glutamate is not removed promptly in the synaptic cleft, it will excessively stimulate the glutamate receptors and induce excitotoxic effects on the CNS. With lack of extracellular enzyme to decompose glutamate, glutamate uptake in the synaptic cleft is mainly achieved by the excitatory amino acid transporters (EAATs, also known as high-affinity glutamate transporters). Current studies have confirmed that decreased expression and function of EAATs appear in PD animal models. Moreover, single unilateral administration of EAATs inhibitor in the substantia nigra mimics several PD features and this is a solid evidence supporting that decreased EAATs contribute to the process of PD. Drugs or treatments promoting the expression and function of EAATs are shown to attenuate dopamine neurons death in the substantia nigra and striatum, ameliorate the behavior disorder, and improve cognitive abilities in PD animal models. EAATs are potential effective drug targets in treatment of PD and thus study of relationship between EAATs and PD has predominant medical significance currently.

EAAT1 is involved in transport ofl-glutamate during differentiation of the Caco-2 cell line

2000 ◽  
Vol 279 (2) ◽  
pp. G366-G373 ◽  
Author(s):  
Agnès Mordrelle ◽  
Eric Jullian ◽  
Cyrille Costa ◽  
Estelle Cormet-Boyaka ◽  
Robert Benamouzig ◽  
...  
Keyword(s):  
Amino Acid ◽  
Cell Line ◽  
Excitatory Amino Acid ◽  
Kinetic Studies ◽  
Glutamate Transport ◽  
Hill Coefficient ◽  
Amino Acid Transporters ◽  
Intestinal Epithelial ◽  
Transport Capacity ◽  
Excitatory Amino Acid Transporter

Little is known concerning the expression of amino acid transporters during intestinal epithelial cell differentiation. The transport mechanism ofl-glutamate and its regulation during the differentiation process were investigated using the human intestinal Caco-2 cell line. Kinetic studies demonstrated the presence of a single, high-affinity,d-aspartate-sensitive l-glutamate transport system in both confluent and fully differentiated Caco-2 cells. This transport was clearly Na+ dependent, with a Hill coefficient of 2.9 ± 0.3, suggesting a 3 Na+-to-1 glutamate stoichiometry and corresponding to the well-characterized XA,G − system. The excitatory amino acid transporter (EAAT)1 transcript was consistently expressed in the Caco-2 cell line, whereas the epithelial and neuronal EAAT3 transporter was barely detected. In contrast with systems B0 and y+, which have previously been reported to be downregulated when Caco-2 cells stop proliferating, l-glutamate transport capacity was found to increase steadily between day 8 and day 17. This increase was correlated with the level of EAAT1 mRNA, which might reflect an increase in EAAT1 gene transcription and/or stabilization of the EAAT1 transcript.

scholarly journals Light-evoked glutamate transporter EAAT5 activation coordinates with conventional feedback inhibition to control rod bipolar cell output

2020 ◽  
Vol 123 (5) ◽  
pp. 1828-1837
Author(s):  
Gregory W. Bligard ◽  
James DeBrecht ◽  
Robert G. Smith ◽  
Peter D. Lukasiewicz
Keyword(s):  
Amino Acid ◽  
Feedback Inhibition ◽  
Excitatory Amino Acid ◽  
Glutamate Transporter ◽  
Bipolar Cells ◽  
Major Contributor ◽  
Excitatory Amino Acid Transporter ◽  
Control Rod ◽  
Neuronal Output ◽  
Rod Bipolar Cells

Excitatory amino acid transporter 5 (EAAT5) glutamate transporters have a chloride channel that is strongly activated by glutamate, which modulates excitatory signaling. We found that EAAT5 is a major contributor to feedback inhibition on rod bipolar cells. Inhibition to rod bipolar cells is also mediated by GABA and glycine. GABA and glycine mediate the early phase of feedback inhibition, and EAAT5 mediates a more delayed inhibition. Together, inhibitory transmitters and EAAT5 coordinate to mediate feedback inhibition, controlling neuronal output.

scholarly journals Modeling of excitatory amino acid transporters and clearance of synaptic cleft on millisecond time scale

2019 ◽  
Vol 14 (4) ◽  
pp. 407
Author(s):  
Denis Shchepakin ◽  
Leonid Kalachev ◽  
Michael Kavanaugh
Keyword(s):  
Amino Acid ◽  
Time Scale ◽  
Time Course ◽  
Transport Model ◽  
Excitatory Amino Acid ◽  
Synaptic Cleft ◽  
Amino Acid Transporters ◽  
Excitatory Amino Acid Transporters ◽  
Millisecond Time Scale ◽  
The Impact

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.

Introduction: Glutamate transport, metabolism, and physiological responses

1999 ◽  
Vol 277 (4) ◽  
pp. F477-F480 ◽  
Author(s):  
M. A. Hediger ◽  
T. C. Welbourne
Keyword(s):  
Amino Acid ◽  
San Francisco ◽  
Excitatory Amino Acid ◽  
Glutamate Transport ◽  
Glutamine Metabolism ◽  
Epithelial Cell Line ◽  
Amino Acid Transporters ◽  
Excitatory Amino Acid Transporter ◽  
Renal Epithelial Cell

The material covered in this set of articles was originally presented at Experimental Biology ’98, in San Francisco, CA, on April 20, 1998. Here, the participants recount important elements of current research on the role of glutamate transporter activity in cellular signaling, metabolism, and organ function. W. A. Fairman and S. G. Amara discuss the five subtypes of human excitatory amino acid transporters, with emphasis on the EAAT4 subtype. M. A. Hediger discusses the expression and action of EAAC1 subtype of the human excitatory amino acid transporter. I. Nissim provides an overview of the significant role of pH in regulating Gln/Glu metabolism in the kidney, liver, and brain. J. D. McGivan and B. Nicholson describe some characteristics of glutamate transport regulation with regard to a specific experimental model of the bovine renal epithelial cell line NBL-1. Finally, T. C. Welbourne and J. C. Matthews introduce the “functional unit” concept of glutamate transport and how this relates to both glutamine metabolism and paracellular permeability.

scholarly journals Up-Regulation of the Excitatory Amino Acid Transporters EAAT1 and EAAT2 by Mammalian Target of Rapamycin

2016 ◽  
Vol 39 (6) ◽  
pp. 2492-2500 ◽  
Author(s):  
Abeer Abousaab ◽  
Nestor Luis Uzcategui ◽  
Bhaeldin Elsir ◽  
Florian Lang
Keyword(s):  
Amino Acid ◽  
Excitatory Amino Acid ◽  
Mammalian Target Of Rapamycin ◽  
Synaptic Cleft ◽  
Target Of Rapamycin ◽  
Amino Acid Transporters ◽  
Protein Abundance ◽  
Excitatory Amino Acid Transporters ◽  
Neuronal Excitation ◽  
Electrode Voltage

Background: The excitatory amino-acid transporters EAAT1 and EAAT2 clear glutamate from the synaptic cleft and thus terminate neuronal excitation. The carriers are subject to regulation by various kinases. The EAAT3 isoform is regulated by mammalian target of rapamycin (mTOR). The present study thus explored whether mTOR influences transport by EAAT1 and/or EAAT2. Methods: cRNA encoding wild type EAAT1 (SLC1A3) or EAAT2 (SLC1A2) was injected into Xenopus oocytes without or with additional injection of cRNA encoding mTOR. Dual electrode voltage clamp was performed in order to determine electrogenic glutamate transport (IEAAT). EAAT2 protein abundance was determined utilizing chemiluminescence. Results: Appreciable IEAAT was observed in EAAT1 or EAAT2 expressing but not in water injected oocytes. IEAAT was significantly increased by coexpression of mTOR. Coexpression of mTOR increased significantly the maximal IEAAT in EAAT1 or EAAT2 expressing oocytes, without significantly modifying affinity of the carriers. Moreover, coexpression of mTOR increased significantly EAAT2 protein abundance in the cell membrane. Conclusions: The kinase mTOR up-regulates the excitatory amino acid transporters EAAT1 and EAAT2.

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