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

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.

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Anion Permeation through Excitatory Amino Acid Transporters

Biophysical Journal ◽

10.1016/j.bpj.2013.11.858 ◽

2014 ◽

Vol 106 (2) ◽

pp. 149a

Author(s):

Jan-Philipp Machtens ◽

Christine Lansche ◽

Ariane Leinenweber ◽

Petra Kilian ◽

Birgit Begemann ◽

...

Keyword(s):

Amino Acid ◽

Excitatory Amino Acid ◽

Amino Acid Transporters ◽

Excitatory Amino Acid Transporters ◽

Anion Permeation

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

Science Advances ◽

10.1126/sciadv.aba9854 ◽

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.

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

Journal of Cerebral Blood Flow & Metabolism ◽

10.1038/jcbfm.2014.30 ◽

2014 ◽

Vol 34 (5) ◽

pp. 764-767 ◽

Cited By ~ 9

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.

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

Cells ◽

10.3390/cells9092037 ◽

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.

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

Journal of Biological Chemistry ◽

10.1074/jbc.ac119.011190 ◽

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.

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Role of EphrinA3 in HIV-1 Neuropathogenesis

ASN NEURO ◽

10.1177/17590914211044359 ◽

2021 ◽

Vol 13 ◽

pp. 175909142110443

Author(s):

Chitra Mohinder Singh Singal ◽

Paritosh Jaiswal ◽

Anuradha Mehta ◽

Kanza Saleem ◽

Pankaj Seth

Keyword(s):

Excitatory Amino Acid ◽

Neuronal Damage ◽

Glutamate Uptake ◽

Synaptic Activity ◽

Glutamate Excitotoxicity ◽

Amino Acid Transporters ◽

Extracellular Glutamate ◽

Hiv 1

Glial cells perform important supporting functions for neurons through a dynamic crosstalk. Neuron–glia communication is the major phenomenon to sustain homeostatic functioning of the brain. Several interactive pathways between neurons and astrocytes are critical for the optimal functioning of neurons, and one such pathway is the ephrinA3–ephA4 signaling. The role of this pathway is essential in maintaining the levels of extracellular glutamate by regulating the excitatory amino acid transporters, EAAT1 and EAAT2 on astrocytes. Human immunodeficiency virus-1 (HIV-1) and its proteins cause glutamate excitotoxicity due to excess glutamate levels at sites of high synaptic activity. This study unravels the effects of HIV-1 transactivator of transcription (Tat) from clade B on ephrinA3 and its role in regulating glutamate levels in astrocyte–neuron co-cultures of human origin. It was observed that the expression of ephrinA3 increases in the presence of HIV-1 Tat B, while the expression of EAAT1 and EAAT2 was attenuated. This led to reduced glutamate uptake and therefore high neuronal death due to glutamate excitotoxicity. Knockdown of ephrinA3 using small interfering RNA, in the presence of HIV-1 Tat B reversed the neurotoxic effects of HIV-1 Tat B via increased expression of glutamate transporters that reduced the levels of extracellular glutamate. The in vitro findings were validated in autopsy brain sections from acquired immunodeficiency syndrome patients and we found ephrinA3 to be upregulated in the case of HIV-1-infected patients. This study offers valuable insights into astrocyte-mediated neuronal damage in HIV-1 neuropathogenesis.

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

Neurobiology of Disease ◽

10.1016/j.nbd.2014.01.011 ◽

2014 ◽

Vol 65 ◽

pp. 69-81 ◽

Cited By ~ 29

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

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The food-associated fungal neurotoxin ochratoxin A inhibits the absorption of glutamate by astrocytes through a decrease in cell surface expression of the excitatory amino-acid transporters GLAST and GLT-1

NeuroToxicology ◽

10.1016/j.neuro.2010.06.003 ◽

2010 ◽

Vol 31 (5) ◽

pp. 475-484 ◽

Cited By ~ 30

Author(s):

Helisoa Razafimanjato ◽

Nicolas Garmy ◽

Xiao-Jun Guo ◽

Karine Varini ◽

Coralie Di Scala ◽

...

Keyword(s):

Amino Acid ◽

Cell Surface ◽

Ochratoxin A ◽

Excitatory Amino Acid ◽

Surface Expression ◽

Cell Surface Expression ◽

Amino Acid Transporters ◽

Excitatory Amino Acid Transporters

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

Neural Plasticity ◽

10.1155/2016/8941327 ◽

2016 ◽

Vol 2016 ◽

pp. 1-8 ◽

Cited By ~ 16

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.

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