scholarly journals Consensus designs and thermal stability determinants of a human glutamate transporter

eLife ◽  
2018 ◽  
Vol 7 ◽  
Author(s):  
Erica Cirri ◽  
Sébastien Brier ◽  
Reda Assal ◽  
Juan Carlos Canul-Tec ◽  
Julia Chamot-Rooke ◽  
...  
Keyword(s):  
Amino Acid ◽  
Molecular Mechanisms ◽  
Excitatory Amino Acid ◽  
Glutamate Transporter ◽  
Amino Acid Identity ◽  
Amino Acid Transporters ◽  
Subunit Interface ◽  
Hydrogen Deuterium ◽  
Excitatory Neurotransmission ◽  
Rate Limit

Human excitatory amino acid transporters (EAATs) take up the neurotransmitter glutamate in the brain and are essential to maintain excitatory neurotransmission. Our understanding of the EAATs’ molecular mechanisms has been hampered by the lack of stability of purified protein samples for biophysical analyses. Here, we present approaches based on consensus mutagenesis to obtain thermostable EAAT1 variants that share up to ~95% amino acid identity with the wild type transporters, and remain natively folded and functional. Structural analyses of EAAT1 and the consensus designs using hydrogen-deuterium exchange linked to mass spectrometry show that small and highly cooperative unfolding events at the inter-subunit interface rate-limit their thermal denaturation, while the transport domain unfolds at a later stage in the unfolding pathway. Our findings provide structural insights into the kinetic stability of human glutamate transporters, and introduce general approaches to extend the lifetime of human membrane proteins for biophysical analyses.

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.

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.

Progressive Parkinsonism by acute dysfunction of excitatory amino acid transporters in the rat substantia nigra

2014 ◽  
Vol 65 ◽  
pp. 69-81 ◽  
Author(s):  
Maxime Assous ◽  
Laurence Had-Aissouni ◽  
Paolo Gubellini ◽  
Christophe Melon ◽  
Imane Nafia ◽  
...  
Keyword(s):  
Amino Acid ◽  
Substantia Nigra ◽  
Excitatory Amino Acid ◽  
Amino Acid Transporters ◽  
Excitatory Amino Acid Transporters ◽  
Rat Substantia Nigra

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.

scholarly journals Klotho Sensitivity of the Neuronal Excitatory Amino Acid Transporters EAAT3 and EAAT4

PLoS ONE ◽  
2013 ◽  
Vol 8 (7) ◽  
pp. e70988 ◽  
Author(s):  
Ahmad Almilaji ◽  
Carlos Munoz ◽  
Tatsiana Pakladok ◽  
Ioana Alesutan ◽  
Martina Feger ◽  
...  
Keyword(s):  
Amino Acid ◽  
Excitatory Amino Acid ◽  
Amino Acid Transporters ◽  
Excitatory Amino Acid Transporters

scholarly journals Interaction of Excitatory Amino Acid Transporters 1 – 3 (EAAT1, EAAT2, EAAT3) with N-Carbamoylglutamate and N-Acetylglutamate

2017 ◽  
Vol 43 (5) ◽  
pp. 1907-1916 ◽  
Author(s):  
Birgitta C. Burckhardt ◽  
Gerhard Burckhardt
Keyword(s):  
Amino Acid ◽  
Excitatory Amino Acid ◽  
Hepatic Uptake ◽  
Xenopus Laevis Oocytes ◽  
Amino Acid Transporters ◽  
Synthetic Analog ◽  
Excitatory Amino Acid Transporters ◽  
Sodium Dependent ◽  
Inward Currents ◽  
Acetylglutamate Synthase

Background/Aims: Inborn deficiency of the N-acetylglutamate synthase (NAGS) impairs the urea cycle and causes neurotoxic hyperammonemia. Oral administration of N-carbamoylglutamate (NCG), a synthetic analog of N-acetylglutamate (NAG), successfully decreases plasma ammonia levels in the affected children. Due to structural similarities to glutamate, NCG may be absorbed in the intestine and taken up into the liver by excitatory amino acid transporters (EAATs). Methods: Using Xenopus laevis oocytes expressing either human EAAT1, 2, or 3, or human sodium-dependent dicarboxylate transporter 3 (NaDC3), transport-associated currents of NAG, NCG, and related dicarboxylates were assayed. Results: L-aspartate and L-glutamate produced saturable inward currents with Km values below 30 µM. Whereas NCG induced a small inward current only in EAAT3 expressing oocytes, NAG was accepted by all EAATs. With EAAT3, the NAG-induced current was sodium-dependent and saturable (Km 409 µM). Oxaloacetate was found as an additional substrate of EAAT3. In NaDC3-expressing oocytes, all dicarboxylates induced much larger inward currents than did L-aspartate and L-glutamate. Conclusion: EAAT3 may contribute to intestinal absorption and hepatic uptake of NCG. With respect to transport of amino acids and dicarboxylates, EAAT3 and NaDC3 can complement each other.

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.

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