scholarly journals Tuning the ion selectivity of glutamate transporter–associated uncoupled conductances

2016 ◽  
Vol 148 (1) ◽  
pp. 13-24 ◽  
Author(s):  
Rosemary J. Cater ◽  
Robert J. Vandenberg ◽  
Renae M. Ryan
Keyword(s):  
Excitatory Amino Acid ◽  
Ion Selectivity ◽  
Glutamate Transporter ◽  
Amino Acid Transporters ◽  
Primary Role ◽  
Cation Selectivity ◽  
Anion Selectivity ◽  
Transport Domain ◽  
Cl Conductance

The concentration of glutamate within a glutamatergic synapse is tightly regulated by excitatory amino acid transporters (EAATs). In addition to their primary role in clearing extracellular glutamate, the EAATs also possess a thermodynamically uncoupled Cl− conductance. This conductance is activated by the binding of substrate and Na+, but the direction of Cl− flux is independent of the rate or direction of substrate transport; thus, the two processes are thermodynamically uncoupled. A recent molecular dynamics study of the archaeal EAAT homologue GltPh (an aspartate transporter from Pyrococcus horikoshii) identified an aqueous pore at the interface of the transport and trimerization domains, through which anions could permeate, and it was suggested that an arginine residue at the most restricted part of this pathway might play a role in determining anion selectivity. In this study, we mutate this arginine to a histidine in the human glutamate transporter EAAT1 and investigate the role of the protonation state of this residue on anion selectivity and transporter function. Our results demonstrate that a positive charge at this position is crucial for determining anion versus cation selectivity of the uncoupled conductance of EAAT1. In addition, because the nature of this residue influences the turnover rate of EAAT1, we reveal an intrinsic link between the elevator movement of the transport domain and the Cl− channel.

scholarly journals A Model for Predicting Cation Selectivity and Permeability in AMPA and NMDA Receptors Based on Receptor Subunit Composition

2021 ◽  
Vol 13 ◽  
Author(s):  
Sampath Kumar ◽  
Sanjay S. Kumar
Keyword(s):  
Experimental Data ◽  
Divalent Cations ◽  
Ion Selectivity ◽  
Gene Families ◽  
Subunit Composition ◽  
Receptor Subunit ◽  
Cation Selectivity ◽  
Subunit Stoichiometry ◽  
Ampa And Nmda Receptors

Glutamatergic AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid) and NMDA (N-methyl-D-aspartate) receptors are implicated in diverse functions ranging from synaptic plasticity to cell death. They are heterotetrameric proteins whose subunits are derived from multiple distinct gene families. The subunit composition of these receptors determines their permeability to monovalent and/or divalent cations, but it is not entirely clear how this selectivity arises in native and recombinantly-expressed receptor populations. By analyzing the sequence of amino acids lining the selectivity filters within the pore forming membrane helices (M2) of these subunits and by correlating subunit stoichiometry of these receptors with their ability to permeate Na+ and/or Ca2+, we propose here a mathematical model for predicting cation selectivity and permeability in these receptors. The model proposed is based on principles of charge attractivity and charge neutralization within the pore forming region of these receptors; it accurately predicts and reconciles experimental data across various platforms including Ca2+ permeability of GluA2-lacking AMPARs and ion selectivity within GluN3-containing di- and tri-heteromeric NMDARs. Additionally, the model provides insights into biophysical mechanisms regulating cation selectivity and permeability of these receptors and the role of various subunits in these processes.

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.

Differential cellular and subcellular distribution of glutamate transporters in the cat retina

2004 ◽  
Vol 21 (4) ◽  
pp. 551-565 ◽  
Author(s):  
BOZENA FYK-KOLODZIEJ ◽  
PU QIN ◽  
ARTURIK DZHAGARYAN ◽  
ROBERTA G. POURCHO
Keyword(s):  
Ganglion Cells ◽  
Excitatory Amino Acid ◽  
Pigment Epithelium ◽  
Glutamate Transporter ◽  
Bipolar Cells ◽  
Amino Acid Transporters ◽  
Cone Photoreceptors ◽  
Glutamatergic Synapses ◽  
Axon Terminals ◽  
Cat Retina

Retrieval of glutamate from extracellular sites in the retina involves at least five excitatory amino acid transporters. Immunocytochemical analysis of the cat retina indicates that each of these transporters exhibits a selective distribution which may reflect its specific function. The uptake of glutamate into Müller cells or astrocytes appears to depend upon GLAST and EAAT4, respectively. Staining for EAAT4 was also seen in the pigment epithelium. The remaining transporters are neuronal with GLT-1α localized to a number of cone bipolar, amacrine, and ganglion cells and GLT-1v in cone photoreceptors and several populations of bipolar cells. The EAAC1 transporter was found in horizontal, amacrine, and ganglion cells. Staining for EAAT5 was seen in the axon terminals of both rod and cone photoreceptors as well as in numerous amacrine and ganglion cells. Although some of the glutamate transporter molecules are positioned for presynaptic or postsynaptic uptake at glutamatergic synapses, others with localizations more distant from such contacts may serve in modulatory roles or provide protection against excitoxic or oxidative damage.

scholarly journals Excitatory amino acid transporters tonically restrain nTS synaptic and neuronal activity to modulate cardiorespiratory function

2016 ◽  
Vol 115 (3) ◽  
pp. 1691-1702 ◽  
Author(s):  
Michael P. Matott ◽  
Brian C. Ruyle ◽  
Eileen M. Hasser ◽  
David D. Kline
Keyword(s):  
Amino Acid ◽  
Excitatory Amino Acid ◽  
Visceral Afferents ◽  
Amino Acid Transporters ◽  
Excitatory Amino Acid Transporters ◽  
Excitatory Neurotransmitter ◽  
Extracellular Milieu ◽  
Cardiorespiratory Function ◽  
The Central Nervous System

The nucleus tractus solitarii (nTS) is the initial central termination site for visceral afferents and is important for modulation and integration of multiple reflexes including cardiorespiratory reflexes. Glutamate is the primary excitatory neurotransmitter in the nTS and is removed from the extracellular milieu by excitatory amino acid transporters (EAATs). The goal of this study was to elucidate the role of EAATs in the nTS on basal synaptic and neuronal function and cardiorespiratory regulation. The majority of glutamate clearance in the central nervous system is believed to be mediated by astrocytic EAAT 1 and 2. We confirmed the presence of EAAT 1 and 2 within the nTS and their colocalization with astrocytic markers. EAAT blockade with dl- threo-β-benzyloxyaspartic acid (TBOA) produced a concentration-related depolarization, increased spontaneous excitatory postsynaptic current (EPSC) frequency, and enhanced action potential discharge in nTS neurons. Solitary tract-evoked EPSCs were significantly reduced by EAAT blockade. Microinjection of TBOA into the nTS of anesthetized rats induced apneic, sympathoinhibitory, depressor, and bradycardic responses. These effects mimicked the response to microinjection of exogenous glutamate, and glutamate responses were enhanced by EAAT blockade. Together these data indicate that EAATs tonically restrain nTS excitability to modulate cardiorespiratory function.

scholarly journals The Ion Permeability Induced in Thin Lipid Membranes by the Polyene Antibiotics Nystatin and Amphotericin B

1970 ◽  
Vol 56 (1) ◽  
pp. 100-124 ◽  
Author(s):  
Albert Cass ◽  
Alan Finkelstein ◽  
Vivian Krespi
Keyword(s):  
Amphotericin B ◽  
Lipid Membranes ◽  
Ion Selectivity ◽  
Membrane Conductance ◽  
Temperature Sensitive ◽  
Hydroxyl Groups ◽  
Large Power ◽  
Polyene Antibiotics ◽  
Cation Selectivity ◽  
Anion Selectivity

Characteristics of nystatin and amphotericin B action on thin (<100 A) lipid membranes are: (a) micromolar amounts increase membrane conductance from 10-8 to over 10-2 Ω-1 cm-2; (b) such membranes are (non-ideally) anion selective and discriminate among anions on the basis of size; (c) membrane sterol is required for action; (d) antibiotic presence on both sides of membrane strongly favors action; (e) conductance is proportional to a large power of antibiotic concentration; (f) conductance decreases ∼104 times for a 10°C temperature rise; (g) kinetics of antibiotic action are also very temperature sensitive; (h) ion selectivity is pH independent between 3 and 10, but (i) activity is reversibly lost at high pH; (j) methyl ester derivatives are fully active; N-acetyl and N-succinyl derivatives are inactive; (k) current-voltage characteristic is nonlinear when membrane separates nonidentical salt solutions. These characteristics are contrasted with those of valinomycin. Observations (a)–(g) suggest that aggregates of polyene and sterol from opposite sides of the membrane interact to create aqueous pores; these pores are not static, but break up (melt) and reform continuously. Mechanism of anion selectivity is obscure. Observations (h)–(j) suggest—NH3+ is important for activity; it is probably not responsible for selectivity, particularly since four polyene antibiotics, each containing two—NH3+ groups, induce ideal cation selectivity. Possibly the many hydroxyl groups in nystatin and amphotericin B are responsible for anion selectivity. The effects of polyene antibiotics on thin lipid membranes are consistent with their action on biological membranes.

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.

Enhancement of substrate-gated Cl− currents via rat glutamate transporter EAAT4 by PMA

2006 ◽  
Vol 290 (5) ◽  
pp. C1334-C1340 ◽  
Author(s):  
Hongyu Fang ◽  
Yueming Huang ◽  
Zhiyi Zuo
Keyword(s):  
Neuronal Excitability ◽  
Excitatory Amino Acid ◽  
Glutamate Transporter ◽  
Reversal Potential ◽  
Chelating Agent ◽  
Amino Acid Transporters ◽  
Transport Activity ◽  
High Concentration ◽  
Excitatory Neurotransmitter ◽  
Induced Currents

Glutamate transporters (also called excitatory amino acid transporters, EAAT) are important in extracellular homeostasis of glutamate, a major excitatory neurotransmitter. EAAT4, a neuronally expressed EAAT in cerebellum, has a large portion (∼95% of the total l-aspartate-induced currents in human EAAT4) of substrate-gated Cl− currents, a distinct feature of this EAAT. We cloned EAAT4 from rat cerebellum. This molecule was predicted to have eight putative transmembrane domains. l-Glutamate induced an inward current in oocytes expressing this EAAT4 at a holding potential −60 mV. Phorbol 12-myristate 13-acetate (PMA), a protein kinase C (PKC) activator, significantly increased the magnitude of l-glutamate-induced currents but did not affect the apparent affinity of EAAT4 for l-glutamate. This PMA-enhanced current had a reversal potential −17 mV at extracellular Cl− concentration ([Cl−]o) 104 mM with an ∼60-mV shift per 10-fold change in [Cl−]o, properties consistent with Cl−-selective conductance. However, PMA did not change EAAT4 transport activity as measured by [3H]-l-glutamate. Thus PMA-enhanced Cl− currents via EAAT4 were not thermodynamically coupled to substrate transport. These PMA-enhanced Cl− currents were partially blocked by staurosporine, chelerythrine, and calphostin C, the three PKC inhibitors. Ro-31-8425, a PKC inhibitor that inhibits conventional PKC isozymes at low concentrations (nM level), partially inhibited the PMA-enhanced Cl− currents only at a high concentration (1 μM). Intracellular injection of BAPTA, a Ca2+-chelating agent, did not affect the PMA-enhanced Cl− currents. 4α-Phorbol-12,13-didecanoate, an inactive analog of PMA, did not enhance glutamate-induced currents. These data suggest that PKC, possibly isozymes other than conventional ones, modulates the substrate-gated Cl− currents via rat EAAT4. Our results also suggest that substrate-gated ion channel activity and glutamate transport activity, two EAAT4 properties that could modulate neuronal excitability, can be regulated independently.

scholarly journals Glutamate transporter dysfunction associated with nerve injury-induced pain in mice

2012 ◽  
Vol 107 (2) ◽  
pp. 649-657 ◽  
Author(s):  
Ian A. Napier ◽  
Sarasa A. Mohammadi ◽  
MacDonald J. Christie
Keyword(s):  
Spinal Cord ◽  
Neuropathic Pain ◽  
Dorsal Horn ◽  
Nerve Injury ◽  
Excitatory Amino Acid ◽  
Expression Patterns ◽  
Glutamate Transporter ◽  
Synaptic Activity ◽  
Amino Acid Transporters ◽  
Functional Changes

Dysfunction at glutamatergic synapses has been proposed as a mechanism in the development of neuropathic pain. Here we sought to determine whether peripheral nerve injury-induced neuropathic pain results in functional changes to primary afferent synapses. Signs of neuropathic pain as well as an induction of glial fibrillary acidic protein in immunostained spinal cord sections 4 days after partial ligation of the sciatic nerve indicated the induction of neuropathic pain. We found that following nerve injury, no discernable change to kinetics of dl-α-amino-3-hydroxy-5-methylisoxazole-propionic acid (AMPA) or N-methyl-d-aspartate receptor (NMDAR)-mediated evoked excitatory postsynaptic currents (eEPSCs) could be observed in dorsal horn (lamina I/II) neurons compared with those of naïve mice. However, we did find that nerve injury was accompanied by slowed decay of the early phase of eEPSCs in the presence of glutamate transporter inhibition by the competitive nontransportable inhibitor dl-threo-β-benzyloxyaspartic acid (TBOA). Concomitantly, expression patterns for the two major glutamate transporters in the spinal cord, excitatory amino acid transporters (EAAT) 1 and EAAT2, were found to be reduced at this time (4 days postinjury). We then sought to directly determine whether nerve injury results in glutamate spillover to NMDARs at dorsal horn synapses. By employing the use-dependent NMDAR blocker (±)MK-801 to block subsynaptic receptors, we found that although TBOA-induced spillover to extrasynaptic receptors trended to increased activation of these receptors after nerve injury, this was not significant compared with naïve mice. Together, these results suggest the development of neuropathic pain involves subtle changes to glutamate transporter expression and function that could contribute to neuropathic pain during excessive synaptic activity.

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 Glutamic Acid Transporters: Targets for Neuroprotective Therapies in Parkinson’s Disease

2021 ◽  
Vol 15 ◽  
Author(s):  
Xiang Li ◽  
Wenjun Wang ◽  
Jianghong Yan ◽  
Fancai Zeng
Keyword(s):  
Parkinson’S Disease ◽  
Parkinson's Disease ◽  
Glutamic Acid ◽  
Drug Targets ◽  
Excitatory Amino Acid ◽  
Glutamate Transporter ◽  
Glutamate Transporters ◽  
Symptomatic Treatment ◽  
Amino Acid Transporters ◽  
Functional Roles

Parkinson’s disease (PD) is a common neurodegenerative disease in middle-aged and elderly individuals. At present, no effective drug has been developed to treat PD. Although a variety of drugs exist for the symptomatic treatment of PD, they all have strong side effects. Most studies on PD mainly focus on dopaminergic neurons. This review highlights the function of glutamic acid transporters (GLTs), including excitatory amino acid transporters (EAATs) and vesicular glutamate transporters (VGLUTs), during the development of PD. In addition, using bioinformatics, we compared the expression of different types of glutamate transporter genes in the cingulate gyrus of PD patients and healthy controls. More importantly, we suggest that the functional roles of glutamate transporters may prove beneficial in the treatment of PD. In summary, VGLUTs and EAATs may be potential targets in the treatment of PD. VGLUTs and EAATs can be used as clinical drug targets to achieve better efficacy. Through this review article, we hope to enable future researchers to improve the condition of PD patients.

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