scholarly journals Na+-dependent gate dynamics and electrostatic attraction ensure substrate coupling in glutamate transporters

2020 ◽  
Vol 6 (47) ◽  
pp. eaba9854
Author(s):  
C. Alleva ◽  
K. Kovalev ◽  
R. Astashkin ◽  
M. I. Berndt ◽  
C. Baeken ◽  
...  
Keyword(s):  
Excitatory Amino Acid ◽  
Glutamate Uptake ◽  
Substrate Binding ◽  
Synaptic Cleft ◽  
Electrostatic Attraction ◽  
Cubic Phase ◽  
Amino Acid Transporters ◽  
X Ray ◽  
Gate Opening ◽  
Substrate Coupling

Excitatory amino acid transporters (EAATs) harness [Na+], [K+], and [H+] gradients for fast and efficient glutamate removal from the synaptic cleft. Since each glutamate is cotransported with three Na+ ions, [Na+] gradients are the predominant driving force for glutamate uptake. We combined all-atom molecular dynamics simulations, fluorescence spectroscopy, and x-ray crystallography to study Na+:substrate coupling in the EAAT homolog GltPh. A lipidic cubic phase x-ray crystal structure of wild-type, Na+-only bound GltPh at 2.5-Å resolution revealed the fully open, outward-facing state primed for subsequent substrate binding. Simulations and kinetic experiments established that only the binding of two Na+ ions to the Na1 and Na3 sites ensures complete HP2 gate opening via a conformational selection-like mechanism and enables high-affinity substrate binding via electrostatic attraction. The combination of Na+-stabilized gate opening and electrostatic coupling of aspartate to Na+ binding provides a constant Na+:substrate transport stoichiometry over a broad range of neurotransmitter concentrations.

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 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.

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.

scholarly journals On the Role of a Conserved Methionine in the Na+-Coupling Mechanism of a Neurotransmitter Transporter Homolog

2021 ◽  
Author(s):  
Wenchang Zhou ◽  
Gianluca Trinco ◽  
Dirk J. Slotboom ◽  
Lucy R. Forrest ◽  
José D. Faraldo-Gómez
Keyword(s):  
Turnover Rate ◽  
Transmembrane Domain ◽  
Excitatory Amino Acid ◽  
Synaptic Cleft ◽  
Underlying Mechanism ◽  
Amino Acid Transporters ◽  
Coupling Mechanism ◽  
Transport Reaction ◽  
Structural Work

AbstractExcitatory amino acid transporters (EAAT) play a key role in glutamatergic synaptic communication. Driven by transmembrane cation gradients, these transporters catalyze the reuptake of glutamate from the synaptic cleft once this neurotransmitter has been utilized for signaling. Two decades ago, pioneering studies in the Kanner lab identified a conserved methionine within the transmembrane domain as key for substrate turnover rate and specificity; later structural work, particularly for the prokaryotic homologs GltPh and GltTk, revealed that this methionine is involved in the coordination of one of the three Na+ ions that are co-transported with the substrate. Albeit extremely atypical, the existence of this interaction is consistent with biophysical analyses of GltPh showing that mutations of this methionine diminish the binding cooperativity between substrates and Na+. It has been unclear, however, whether this intriguing methionine influences the thermodynamics of the transport reaction, i.e., its substrate:ion stoichiometry, or whether it simply fosters a specific kinetics in the binding reaction, which, while influential for the turnover rate, do not fundamentally explain the ion-coupling mechanism of this class of transporters. Here, studies of GltTk using experimental and computational methods independently arrive at the conclusion that the latter hypothesis is the most plausible, and lay the groundwork for future efforts to uncover the underlying mechanism.

scholarly journals Cellular Physiology and Pathophysiology of EAAT Anion Channels

2022 ◽  
Vol 15 ◽  
Author(s):  
Peter Kovermann ◽  
Miriam Engels ◽  
Frank Müller ◽  
Christoph Fahlke
Keyword(s):  
Energy Demand ◽  
Excitatory Amino Acid ◽  
High Capacity ◽  
Synaptic Cleft ◽  
Glutamate Transport ◽  
Cell Physiology ◽  
Amino Acid Transporters ◽  
Anion Channels ◽  
Human Genetic Disease ◽  
Experimental Approaches

Excitatory amino acid transporters (EAATs) optimize the temporal resolution and energy demand of mammalian excitatory synapses by quickly removing glutamate from the synaptic cleft into surrounding neuronal and glial cells and ensuring low resting glutamate concentrations. In addition to secondary active glutamate transport, EAATs also function as anion channels. The channel function of these transporters is conserved in all homologs ranging from archaebacteria to mammals; however, its physiological roles are insufficiently understood. There are five human EAATs, which differ in their glutamate transport rates. Until recently the high-capacity transporters EAAT1, EAAT2, and EAAT3 were believed to conduct only negligible anion currents, with no obvious function in cell physiology. In contrast, the low-capacity glutamate transporters EAAT4 and EAAT5 are thought to regulate neuronal signaling as glutamate-gated channels. In recent years, new experimental approaches and novel animal models, together with the discovery of a human genetic disease caused by gain-of-function mutations in EAAT anion channels have enabled identification of the first physiological and pathophysiological roles of EAAT anion channels.

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 The Efficiency of Glutamate Uptake Differs between Neonatal and Adult Cortical Microvascular Endothelial Cells

2014 ◽  
Vol 34 (5) ◽  
pp. 764-767 ◽  
Author(s):  
Maryline Lecointre ◽  
Michelle Hauchecorne ◽  
Armelle Chaussivert ◽  
Stéphane Marret ◽  
Philippe Leroux ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Excitatory Amino Acid ◽  
Glutamate Uptake ◽  
Adult Brain ◽  
Amino Acid Transporters ◽  
Brain Endothelial Cells ◽  
Immature Brain ◽  
Differential Expression Pattern ◽  
Glutamate Efflux ◽  
Brain Homeostasis

Glutamate transporters (excitatory amino-acid transporters (EAATs)) are essential for brain homeostasis. While previous studies indicate that the vascular endothelium contributes to glutamate efflux in the adult brain, little information is available regarding glutamate uptake in the immature brain. The present study shows a differential expression pattern of EAATs between cortical microvessels in adults and newborns. In addition, adult cortical endothelial cells take up glutamate more efficiently than neonatal cells. Our findings indicate age-specific changes in extracellular glutamate regulation by brain endothelial cells, suggesting differences in the efficiency of glutamate efflux during an excitotoxic process that, in turn, may contribute to age-specific brain vulnerability.

scholarly journals Substrate transport and anion permeation proceed through distinct pathways in glutamate transporters

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Mary Hongying Cheng ◽  
Delany Torres-Salazar ◽  
Aneysis D Gonzalez-Suarez ◽  
Susan G Amara ◽  
Ivet Bahar
Keyword(s):  
Excitatory Amino Acid ◽  
Glutamate Uptake ◽  
Amino Acid Transporters ◽  
Structural Coupling ◽  
Substrate Transport ◽  
Hydrophobic Residues ◽  
Substituted Cysteine Accessibility ◽  
Anion Permeation ◽  
Binding Core ◽  
Flexible Wall

Advances in structure-function analyses and computational biology have enabled a deeper understanding of how excitatory amino acid transporters (EAATs) mediate chloride permeation and substrate transport. However, the mechanism of structural coupling between these functions remains to be established. Using a combination of molecular modeling, substituted cysteine accessibility, electrophysiology and glutamate uptake assays, we identified a chloride-channeling conformer, iChS, transiently accessible as EAAT1 reconfigures from substrate/ion-loaded into a substrate-releasing conformer. Opening of the anion permeation path in this iChS is controlled by the elevator-like movement of the substrate-binding core, along with its wall that simultaneously lines the anion permeation path (global); and repacking of a cluster of hydrophobic residues near the extracellular vestibule (local). Moreover, our results demonstrate that stabilization of iChS by chemical modifications favors anion channeling at the expense of substrate transport, suggesting a mutually exclusive regulation mediated by the movement of the flexible wall lining the two regions.

scholarly journals Gateways for Glutamate Neuroprotection in Parkinson’s Disease (PD): Essential Role of EAAT3 and NCX1 Revealed in an In Vitro Model of PD

Cells ◽  
2020 ◽  
Vol 9 (9) ◽  
pp. 2037
Author(s):  
Silvia Piccirillo ◽  
Simona Magi ◽  
Alessandra Preziuso ◽  
Pasqualina Castaldo ◽  
Salvatore Amoroso ◽  
...  
Keyword(s):  
Parkinson’S Disease ◽  
Parkinson's Disease ◽  
Ischemia Reperfusion Injury ◽  
Excitatory Amino Acid ◽  
Cell Damage ◽  
Glutamate Uptake ◽  
Ischemia Reperfusion ◽  
Amino Acid Transporters ◽  
Mitochondrial Functions

Increasing evidence suggests that metabolic alterations may be etiologically linked to neurodegenerative disorders such as Parkinson’s disease (PD) and in particular empathizes the possibility of targeting mitochondrial dysfunctions to improve PD progression. Under different pathological conditions (i.e., cardiac and neuronal ischemia/reperfusion injury), we showed that supplementation of energetic substrates like glutamate exerts a protective role by preserving mitochondrial functions and enhancing ATP synthesis through a mechanism involving the Na+-dependent excitatory amino acid transporters (EAATs) and the Na+/Ca2+ exchanger (NCX). In this study, we investigated whether a similar approach aimed at promoting glutamate metabolism would be also beneficial against cell damage in an in vitro PD-like model. In retinoic acid (RA)-differentiated SH-SY5Y cells challenged with α-synuclein (α-syn) plus rotenone (Rot), glutamate significantly improved cell viability by increasing ATP levels, reducing oxidative damage and cytosolic and mitochondrial Ca2+ overload. Glutamate benefits were strikingly lost when either EAAT3 or NCX1 expression was knocked down by RNA silencing. Overall, our results open the possibility of targeting EAAT3/NCX1 functions to limit PD pathology by simultaneously favoring glutamate uptake and metabolic use in dopaminergic neurons.

scholarly journals Millisecond dynamics of an unlabeled amino acid transporter

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Tina R. Matin ◽  
George R. Heath ◽  
Gerard H. M. Huysmans ◽  
Olga Boudker ◽  
Simon Scheuring
Keyword(s):  
Amino Acid ◽  
Temporal Resolution ◽  
High Speed ◽  
Excitatory Amino Acid ◽  
Synaptic Cleft ◽  
Amino Acid Transporters ◽  
Transition Analysis ◽  
Force Microscopy ◽  
Transport Dynamics ◽  
Physiological Processes

Abstract Excitatory amino acid transporters (EAATs) are important in many physiological processes and crucial for the removal of excitatory amino acids from the synaptic cleft. Here, we develop and apply high-speed atomic force microscopy line-scanning (HS-AFM-LS) combined with automated state assignment and transition analysis for the determination of transport dynamics of unlabeled membrane-reconstituted GltPh, a prokaryotic EAAT homologue, with millisecond temporal resolution. We find that GltPh transporters can operate much faster than previously reported, with state dwell-times in the 50 ms range, and report the kinetics of an intermediate transport state with height between the outward- and inward-facing states. Transport domains stochastically probe transmembrane motion, and reversible unsuccessful excursions to the intermediate state occur. The presented approach and analysis methodology are generally applicable to study transporter kinetics at system-relevant temporal resolution.

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