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.

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.

Water and urea permeation pathways of the human excitatory amino acid transporter EAAT1

2011 ◽  
Vol 439 (2) ◽  
pp. 333-340 ◽  
Author(s):  
Robert J. Vandenberg ◽  
Cheryl A. Handford ◽  
Ewan M. Campbell ◽  
Renae M. Ryan ◽  
Andrea J. Yool
Keyword(s):  
Amino Acid ◽  
Water Transport ◽  
Transport Process ◽  
Excitatory Amino Acid ◽  
Anion Channel ◽  
Glutamate Transporters ◽  
Amino Acid Transporter ◽  
Glutamate Transport ◽  
Excitatory Amino Acid Transporter ◽  
Permeation Properties

Glutamate transport is coupled to the co-transport of 3 Na+ and 1 H+ followed by the counter-transport of 1 K+. In addition, glutamate and Na+ binding to glutamate transporters generates an uncoupled anion conductance. The human glial glutamate transporter EAAT1 (excitatory amino acid transporter 1) also allows significant passive and active water transport, which suggests that water permeation through glutamate transporters may play an important role in glial cell homoeostasis. Urea also permeates EAAT1 and has been used to characterize the permeation properties of the transporter. We have previously identified a series of mutations that differentially affect either the glutamate transport process or the substrate-activated channel function of EAAT1. The water and urea permeation properties of wild-type EAAT1 and two mutant transporters were measured to identify which permeation pathway facilitates the movement of these molecules. We demonstrate that there is a significant rate of L-glutamate-stimulated passive and active water transport. Both the passive and active L-glutamate-stimulated water transport is most closely associated with the glutamate transport process. In contrast, L-glutamate-stimulated [14C]urea permeation is associated with the anion channel of the transporter. However, there is also likely to be a transporter-specific, but glutamate independent, flux of water via the anion channel.

scholarly journals Molecular Basis of Coupled Transport and Anion Conduction in Excitatory Amino Acid Transporters

2021 ◽  
Author(s):  
Claudia Alleva ◽  
Jan-Philipp Machtens ◽  
Daniel Kortzak ◽  
Ingo Weyand ◽  
Christoph Fahlke
Keyword(s):  
Amino Acid ◽  
Molecular Mechanisms ◽  
Excitatory Amino Acid ◽  
Glutamate Transporters ◽  
Glutamate Transport ◽  
Amino Acid Transporters ◽  
Processing Unit ◽  
Coupled Transport ◽  
Excitatory Amino Acid Transporters ◽  
Anion Conduction

AbstractGlutamate is the major excitatory neurotransmitter in the mammalian central nervous system. After its release from presynaptic nerve terminals, glutamate is quickly removed from the synaptic cleft by excitatory amino acid transporters (EAATs) 1–5, a subfamily of glutamate transporters. The five proteins utilize a complex transport stoichiometry that couples glutamate transport to the symport of three Na+ ions and one H+ in exchange with one K+ to accumulate glutamate against up to 106-fold concentration gradients. They are also anion-selective channels that open and close during transitions along the glutamate transport cycle. EAATs belong to a larger family of secondary-active transporters, the SLC1 family, which also includes purely Na+- or H+-coupled prokaryotic transporters and Na+-dependent neutral amino acid exchangers. In recent years, molecular cloning, heterologous expression, cellular electrophysiology, fluorescence spectroscopy, structural approaches, and molecular simulations have uncovered the molecular mechanisms of coupled transport, substrate selectivity, and anion conduction in EAAT glutamate transporters. Here we review recent findings on EAAT transport mechanisms, with special emphasis on the highly conserved hairpin 2 gate, which has emerged as the central processing unit in many of these functions.

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 A computational approach yields selective inhibitors of human excitatory amino acid transporter 2 (EAAT2)

2020 ◽  
Vol 295 (13) ◽  
pp. 4359-4366
Author(s):  
Kelly L. Damm-Ganamet ◽  
Marie-Laure Rives ◽  
Alan D. Wickenden ◽  
Heather M. McAllister ◽  
Taraneh Mirzadegan
Keyword(s):  
Amino Acid ◽  
Drug Target ◽  
Therapeutic Potential ◽  
Excitatory Amino Acid ◽  
Glutamate Uptake ◽  
Homology Model ◽  
Binding Pocket ◽  
Amino Acid Transporters ◽  
Excitatory Amino Acid Transporter ◽  
Significant Interest

Excitatory amino acid transporters (EAATs) represent a protein family that is an emerging drug target with great therapeutic potential for managing central nervous system disorders characterized by dysregulation of glutamatergic neurotransmission. As such, it is of significant interest to discover selective modulators of EAAT2 function. Here, we applied computational methods to identify specific EAAT2 inhibitors. Utilizing a homology model of human EAAT2, we identified a binding pocket at the interface of the transport and trimerization domain. We next conducted a high-throughput virtual screen against this site and identified a selective class of EAAT2 inhibitors that were tested in glutamate uptake and whole-cell electrophysiology assays. These compounds represent potentially useful pharmacological tools suitable for further exploration of the therapeutic potential of EAAT2 and may provide molecular insights into mechanisms of allosteric modulation for glutamate transporters.

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.

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