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 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.

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.

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 Ataxia-linked SLC1A3 mutations alter EAAT1 chloride channel activity and glial regulation of CNS function

2021 ◽  
Author(s):  
Qianyi Wu ◽  
Azman Akhter ◽  
Shashank Pant ◽  
Eunjoo Cho ◽  
Jin Xin Zhu ◽  
...  
Keyword(s):  
Glial Cells ◽  
Excitatory Amino Acid ◽  
Physiological Role ◽  
Glutamate Transport ◽  
Xenopus Laevis Oocytes ◽  
Amino Acid Transporters ◽  
Channel Activity ◽  
Excitatory Neurotransmitter ◽  
Cl Channel ◽  
Cl Channels

Glutamate is the predominant excitatory neurotransmitter in the mammalian central nervous system (CNS). Excitatory Amino Acid Transporters (EAATs) regulate extracellular glutamate by transporting it into cells, mostly glia, to terminate neurotransmission and to avoid neurotoxicity. EAATs are also chloride (Cl-) channels, but the physiological role of Cl- conductance through EAATs is poorly understood. Mutations of human EAAT1 (hEAAT1) have been identified in patients with episodic ataxia type 6 (EA6). One mutation showed increased Cl- channel activity and decreased glutamate transport, but the relative contributions of each function of hEAAT1 to mechanisms underlying the pathology of EA6 remain unclear. Here we investigated the effects of five additional EA6-related mutations on hEAAT1 function in Xenopus laevis oocytes, and on CNS function in a Drosophila melanogaster model of locomotor behavior. Our results indicate that mutations with decreased hEAAT1 Cl- channel activity and functional glutamate transport can also contribute to the pathology of EA6, highlighting the importance of Cl- homeostasis in glial cells for proper CNS function. We also identified a novel mechanism involving an ectopic sodium (Na+) leak conductance in glial cells. Together, these results strongly support the idea that EA6 is primarily an ion channelopathy of CNS glia.

scholarly journals Loss of the glial glutamate transporter eaat2a leads to a combined developmental and epileptic encephalopathy in zebrafish

2021 ◽  
Author(s):  
Adriana L. Hotz ◽  
Ahmed Jamali ◽  
Nicolas N. Rieser ◽  
Stephanie Niklaus ◽  
Ecem Aydin ◽  
...  
Keyword(s):  
Neuronal Excitability ◽  
Excitatory Amino Acid ◽  
Brain Activity ◽  
Glutamate Transporter ◽  
Epileptic Seizures ◽  
Synaptic Cleft ◽  
Epileptic Encephalopathy ◽  
Network Activity ◽  
Excitatory Amino Acid Transporter ◽  
The Impact

ABSTRACTAstroglial excitatory amino acid transporter 2 (EAAT2, GLT-1, SLC1A2) regulates the duration and extent of neuronal excitation by removing glutamate from the synaptic cleft. Human patients with altered EAAT2 function exhibit epileptic seizures, suggesting an important role for astroglial glutamate transporters in balancing neuronal excitability. To study the impact of EAAT2 function at the neural network levels, we generated eaat2a mutant zebrafish. We observed that eaat2a-/- mutant zebrafish larvae display recurrent spontaneous and light-induced seizures in neurons and astroglia, which coincide with an abrupt increase in extracellular glutamate levels. In stark contrast to this hyperexcitability, basal brain activity was surprisingly reduced in eaat2a-/- mutant animals, which manifested in decreased locomotion, neuronal and astroglial calcium signals. Our results reveal an unexpected key role of the astroglial EAAT2a in balancing brain excitability, affecting both neuronal and astroglial network activity.

Functional diversity of excitatory amino acid transporters: ion channel and transport modes

1999 ◽  
Vol 277 (4) ◽  
pp. F481-F486 ◽  
Author(s):  
W. A. Fairman ◽  
S. G. Amara
Keyword(s):  
Central Nervous System ◽  
Arachidonic Acid ◽  
Amino Acid ◽  
Neuronal Excitability ◽  
Excitatory Amino Acid ◽  
Glutamate Transporters ◽  
Amino Acid Transporters ◽  
Proton Conductance ◽  
Cellular Processes ◽  
The Central Nervous System

Recent studies of glutamate transporters in the central nervous system indicate that in addition to their fundamental role in mediating neurotransmitter uptake, these proteins may contribute to the modulation of a variety of cellular processes. Activation of the excitatory amino acid (EAA) carriers generates an electrogenic current attibutable to ion-coupled cotransport. In addition to this transport-associated current, a substrate-gated thermodynamically uncoupled anion flux has been identified that has been proposed to dampen neuronal excitability. Arachidonic acid has been reported to modulate a variety of membrane proteins involved in cellular signaling. Here we discuss recent findings that indicate arachidonic acid stimulates a previously uncharacterized proton-selective conductance in the Purkinje cell-specific subtype, EAAT4. The unique channel-like porperties of the EAATs, their unexpected localization, and physiological evidence propose a modulatory role for the EAATs in neuronal signaling and suggest a broader role for glutamate transporters than simply the clearance of synaptically released glutamate. Thus, the identification of this arachidonate-stimulated proton conductance extends the complexity of mechanisms through which glutamate transporters modulate neuronal excitability.

scholarly journals Excitatory Amino Acid Transporters in Physiology and Disorders of the Central Nervous System

2019 ◽  
Vol 20 (22) ◽  
pp. 5671 ◽  
Author(s):  
Malik ◽  
Willnow
Keyword(s):  
Central Nervous System ◽  
Nervous System ◽  
Amino Acid ◽  
Chloride Channels ◽  
Excitatory Amino Acid ◽  
Amino Acid Transporters ◽  
Excitatory Amino Acid Transporters ◽  
Excitatory Neurotransmitter ◽  
Cerebellar Ataxias ◽  
The Central Nervous System

Excitatory amino acid transporters (EAATs) encompass a class of five transporters with distinct expression in neurons and glia of the central nervous system (CNS). EAATs are mainly recognized for their role in uptake of the amino acid glutamate, the major excitatory neurotransmitter. EAATs-mediated clearance of glutamate released by neurons is vital to maintain proper glutamatergic signalling and to prevent toxic accumulation of this amino acid in the extracellular space. In addition, some EAATs also act as chloride channels or mediate the uptake of cysteine, required to produce the reactive oxygen speciesscavenger glutathione. Given their central role in glutamate homeostasis in the brain, as well as their additional activities, it comes as no surprise that EAAT dysfunctions have been implicated in numerous acute or chronic diseases of the CNS, including ischemic stroke and epilepsy, cerebellar ataxias, amyotrophic lateral sclerosis, Alzheimer’s disease and Huntington’s disease. Here we review the studies in cellular and animal models, as well as in humans that highlight the roles of EAATs in the pathogenesis of these devastating disorders. We also discuss the mechanisms regulating EAATs expression and intracellular trafficking and new exciting possibilities to modulate EAATs and to provide neuroprotection in course of pathologies affecting the CNS.

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.

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