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Robert E Mccullumsmith - One of the best experts on this subject based on the ideXlab platform.
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Glutamate Transporter splice variant expression in an enriched pyramidal cell population in schizophrenia
Translational Psychiatry, 2015Co-Authors: Sinead M Odonovan, Kathryn Hasselfeld, Douglas C Bauer, Micah Simmons, Panos Roussos, Vahram Haroutunian, James H Meadorwoodruff, Robert E MccullumsmithAbstract:Dysregulation of the Glutamate Transporters EAAT1 and EAAT2 and their isoforms have been implicated in schizophrenia. EAAT1 and EAAT2 expression has been studied in different brain regions but the prevalence of astrocytic Glutamate Transporter expression masks the more subtle changes in excitatory amino acid Transporters (EAATs) isoforms in neurons in the cortex. Using laser capture microdissection, pyramidal neurons were cut from the anterior cingulate cortex of postmortem schizophrenia (n=20) and control (n=20) subjects. The messenger RNA (mRNA) levels of EAAT1, EAAT2 and the splice variants EAAT1 exon9skipping, EAAT2 exon9skipping and EAAT2b were analyzed by real time PCR (RT-PCR) in an enriched population of neurons. Region-level expression of these transcripts was measured in postmortem schizophrenia (n=25) and controls (n=25). The relationship between selected EAAT polymorphisms and EAAT splice variant expression was also explored. Anterior cingulate cortex pyramidal cell expression of EAAT2b mRNA was increased (P<0.001; 67%) in schizophrenia subjects compared with controls. There was no significant change in other EAAT variants. EAAT2 exon9skipping mRNA was increased (P<0.05; 38%) at region level in the anterior cingulate cortex with no significant change in other EAAT variants at region level. EAAT2 single-nucleotide polymorphisms were significantly associated with changes in EAAT2 isoform expression. Haloperidol decanoate-treated animals, acting as controls for possible antipsychotic effects, did not have significantly altered neuronal EAAT2b mRNA levels. The novel finding that EAAT2b levels are increased in populations of anterior cingulate cortex pyramidal cells further demonstrates a role for neuronal Glutamate Transporter splice variant expression in schizophrenia.
Reinhard Jahn - One of the best experts on this subject based on the ideXlab platform.
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molecular cloning and functional characterization of human vesicular Glutamate Transporter 3
EMBO Reports, 2002Co-Authors: Shigeo Takamori, Pari Malherbe, Clemens Broger, Reinhard JahnAbstract:Glutamate is the major excitatory neurotransmitter in the mammalian CNS. It is loaded into synaptic vesicles by a proton gradient-dependent uptake system and is released by exocytosis upon stimulation. Recently, two mammalian isoforms of a vesicular Glutamate Transporter, VGLUT1 and VGLUT2, have been identified, the expression of which enables quantal release of Glutamate from Glutamatergic neurons. Here, we report a novel isoform of a human vesicular Glutamate Transporter (hVGLUT3). The predicted amino acid sequence of hVGLUT3 shows 72% identity to both hVGLUT1 and hVGLUT2. hVGLUT3 functions as a vesicular Glutamate Transporter with similar properties to the other isoforms when it is heterologously expressed in a neuroendocrine cell line. Although mammalian VGLUT1 and VGLUT2 exhibit a complementary expression pattern covering all Glutamatergic pathways in the CNS, expression of hVGLUT3 overlaps with them in some brain areas, suggesting molecular diversity that may account for physiological heterogeneity in Glutamatergic synapses.
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identification of differentiation associated brain specific phosphate Transporter as a second vesicular Glutamate Transporter vglut2
The Journal of Neuroscience, 2001Co-Authors: Shigeo Takamori, Jeongseop Rhee, Christian Rosenmund, Reinhard JahnAbstract:Glutamate is the major excitatory neurotransmitter in mammalian CNS. In the presynaptic nerve terminal, Glutamate is stored in synaptic vesicles and released by exocytosis. Previously, it has been shown that a transport protein originally identified as a brain-specific Na-dependent inorganic phosphate Transporter I (BNPI) functions as vesicular Glutamate Transporter and thus has been renamed VGLUT1. Recently, a protein highly homologous to VGLUT1, “differentiation-associated BNPI” (DNPI), has been discovered. Northern blot and in situ hybridization analyses indicate that DNPI mRNA is expressed in some brain regions in which VGLUT1 mRNA is not expressed. We now show that DNPI functions as vesicular Glutamate Transporter with properties very similar to VGLUT1 and propose to rename the protein VGLUT2. VGLUT2 is highly enriched in synaptic vesicles. Furthermore, VGLUT2 resides on a vesicle population that is distinct from vesicles containing the vesicular GABA Transporter or VGLUT1, showing that the expression of VGLUT1 and VGLUT2 do not overlap. When VGLUT2 was expressed in BON cells, membrane fractions displayed ATPdependent, carbonyl cyanide p-trifluoromethoxyphenylhydrazone-sensitive Glutamate uptake. Overexpression of VGLUT2 in cultured autaptic GABAergic neurons yielded postsynaptic currents that were insensitive to the GABAA receptor antagonist bicuculline but blocked by the AMPA-receptor antagonist 2,3dihydroxy-6-nitro-7-sulfonyl-benzo[F]quinoxaline. Thus, expression of VGLUT2 suffices to cause GABAergic neurons to release Glutamate in addition to GABA in a manner very similar to that reported previously for VGLUT1.
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identification of a vesicular Glutamate Transporter that defines a Glutamatergic phenotype in neurons
Nature, 2000Co-Authors: Shigeo Takamori, Jeongseop Rhee, Christian Rosenmund, Reinhard JahnAbstract:Glutamate is the major excitatory neurotransmitter in the mammalian central nervous system. Synaptic vesicles are loaded with neurotransmitter by means of specific vesicular Transporters. Here we show that expression of BNPI, a vesicle-bound Transporter associated with sodium-dependent phosphate transport, results in Glutamate uptake by intracellular vesicles. Substrate specificity and energy dependence are very similar to Glutamate uptake by synaptic vesicles. Stimulation of exocytosis--fusion of the vesicles with the cell membrane and release of their contents--resulted in quantal release of Glutamate from BNPI-expressing cells. Furthermore, we expressed BNPI in neurons containing GABA (gamma-aminobutyric acid) and maintained them as cultures of single, isolated neurons that form synapses to themselves. After stimulation of these neurons, a component of the postsynaptic current is mediated by Glutamate as it is blocked by a combination of the Glutamate receptor antagonists, but is insensitive to a GABA(A) receptor antagonist. We conclude that BNPI functions as vesicular Glutamate Transporter and that expression of BNPI suffices to define a Glutamatergic phenotype in neurons.
Jeffrey D Rothstein - One of the best experts on this subject based on the ideXlab platform.
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reticulon rtn2b regulates trafficking and function of neuronal Glutamate Transporter eaac1
Journal of Biological Chemistry, 2008Co-Authors: Svetlana Vidensky, Alicia M Ruggiero, Susanne Maier, Harald H Sitte, Jeffrey D RothsteinAbstract:Excitatory amino acid Transporters (EAATs) are the primary regulators of extracellular Glutamate concentrations in the central nervous system. Their dysfunction may contribute to several neurological diseases. To date, five distinct mammalian Glutamate Transporters have been cloned. In brain, EAAC1 (excitatory amino acid carrier 1) is the primary neuronal Glutamate Transporter, localized on the perisynaptic membranes that are near release sites. Despite its potential importance in synaptic actions, little is known concerning the regulation of EAAC1 trafficking from the endoplasmic reticulum (ER) to the cell surface. Previously, we identified an EAAC1-associated protein, GTRAP3-18, an ER protein that prevents ER exit of EAAC1 when induced. Here we show that RTN2B, a member of the reticulon protein family that mainly localizes in the ER and ER exit sites interacts with EAAC1 and GTRAP3-18. EAAC1 and GTRAP3-18 bind to different regions of RTN2B. Each protein can separately and independently form complexes with EAAC1. RTN2B enhances ER exit and the cell surface composition of EAAC1 in heterologous cells. Expression of short interfering RNA-mediated knockdown of RTN2B decreases the EAAC1 protein level in neurons. Overall, our results suggest that RTN2B functions as a positive regulator in the delivery of EAAC1 from the ER to the cell surface. These studies indicate that Transporter exit from the ER controlled by the interaction with its ER binding partner represents a critical regulatory step in Glutamate Transporter trafficking to the cell surface.
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Glutamate Transporter expression and function in human glial progenitors.
Glia, 2004Co-Authors: Nicholas J. Maragakis, Joerg Dietrich, Victor W. Wong, Haipeng Xue, Margot Mayer-pröschel, Mahendra S. Rao, Jeffrey D RothsteinAbstract:Glutamate is the major neurotransmitter of the brain, whose extracellular levels are tightly controlled by Glutamate Transporters. Five Glutamate Transporters in the human brain (EAAT1-5) are present on both astroglia and neurons. We characterize the profile of three different human astroglial progenitors in vitro: human glial restricted precursors (HGRP), human astrocyte precursors (HAPC), and early-differentiated astrocytes. EAAT 1, EAAT3, and EAAT4 are all expressed in GRPs with a subsequent upregulation of EAAT1 following differentiation of GRPs into GRP-derived astrocytes in the presence of bone morphogenic protein (BMP-4). This corresponds to a significant increase in the Glutamate transport capacity of these cells. EAAT2, the Transporter responsible for the bulk of Glutamate transport in the adult brain, is not expressed as a full-length protein, nor does it appear to have functional significance (as determined by the EAAT2 inhibitor dihydrokainate) in these precursors. A splice variant of EAAT2, termed EAAT2b, does appear to be present in low levels, however. EAAT3 and EAAT4 expression is reduced as glial maturation progresses both in astrocyte precursors and early-differentiated astrocytes and is consistent with their role in adult tissues as primarily neuronal Glutamate Transporters. These human glial precursors offer several advantages as tools for understanding glial biology because they can be passaged extensively in the presence of mitogens, afford the potential to study the temporal changes in Glutamate Transporter expression in a tightly controlled fashion, and are cultured in the absence of neuronal coculture, allowing for the independent study of astroglial biology.
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Distribution of Glutamate Transporter subtypes during human brain development.
Journal of neurochemistry, 2002Co-Authors: Osnat Bar-peled, Akiko Furuta, Herzel Ben-hur, Anat Biegon, Yoram Groner, Steve Dewhurst, Jeffrey D RothsteinAbstract:In the mature brain, removal of Glutamate from the synaptic cleft plays an important role in the maintenance of subtoxic levels of Glutamate. This requirement is handled by a family of Glutamate Transporters, EAAT1, EAAT2, EAAT3, and EAAT4. Due to the involvement of Glutamate also in neuronal development, it is believed that Glutamate transport plays a role in developmental processes as well. Therefore, we have used immunohistochemical and immunoblot analysis to determine the distribution of the four Glutamate Transporters during human brain development using human pre- and postnatal brain tissue. Regional analysis showed that each Transporter subtype has a unique distribution during development. EAAT2 was the most prominent Glutamate Transporter subtype and was highly enriched in cortex, basal ganglia, cerebellum, and thalamus in all ages examined. EAAT1 immunoreactivity was lower than that of EAAT2, with predominant localization in cortex, basal ganglia, hippocampus, and periventricular region. EAAT3 was located mainly in cortex, basal ganglia, and hippocampus, and EAAT4 was found only in cortex, hippocampus, and cerebellar cortex. The distinct regional distribution of various EAAT subtypes and also the transient expression of specific EAAT subtypes during development suggest multiple functional roles for Glutamate Transporters in the developing brain.
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a neuronal Glutamate Transporter contributes to neurotransmitter gaba synthesis and epilepsy
The Journal of Neuroscience, 2002Co-Authors: Jehuda P Sepkuty, R. Ganel, Akiva S Cohen, Christine U Eccles, Azhar Rafiq, Kevin L Behar, Douglas A Coulter, Jeffrey D RothsteinAbstract:The predominant neuronal Glutamate Transporter, EAAC1 (for excitatory amino acid carrier-1), is localized to the dendrites and somata of many neurons. Rare presynaptic localization is restricted to GABA terminals. Because Glutamate is a precursor for GABA synthesis, we hypothesized that EAAC1 may play a role in regulating GABA synthesis and, thus, could cause epilepsy in rats when inactivated. Reduced expression of EAAC1 by antisense treatment led to behavioral abnormalities, including staring–freezing episodes and electrographic (EEG) seizures. Extracellular hippocampal and thalamocortical slice recordings showed excessive excitability in antisense-treated rats. Patch-clamp recordings of miniature IPSCs (mIPSCs) conducted in CA1 pyramidal neurons in slices from EAAC1 antisense-treated animals demonstrated a significant decrease in mIPSC amplitude, indicating decreased tonic inhibition. There was a 50% loss of hippocampal GABA levels associated with knockdown of EAAC1, and newly synthesized GABA from extracellular Glutamate was significantly impaired by reduction of EAAC1 expression. EAAC1 may participate in normal GABA neurosynthesis and limbic hyperexcitability, whereas epilepsy can result from a disruption of the interaction between EAAC1 and GABA metabolism.
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Modulation of the neuronal Glutamate Transporter EAAT4 by two interacting proteins
Nature, 2001Co-Authors: Mandy Jackson, Wei Song, Mu Ya Liu, Lin Jin, Margaret Dykes-hoberg, Chien Liang G. Lin, William J. Bowers, Howard J. Federoff, Paul C. Sternweis, Jeffrey D RothsteinAbstract:Glutamate is the main excitatory neurotransmitter in the mammalian central nervous system and is removed from the synaptic cleft by sodium-dependent Glutamate Transporters. To date, five distinct Glutamate Transporters have been cloned from animal and human tissue: GLAST (EAAT1), GLT-1 (EAAT2), EAAC1 (EAAT3), EAAT4, and EAAT5 (refs 1,2,3,4,5). GLAST and GLT-1 are localized primarily in astrocytes6,7, whereas EAAC1 (refs 8, 9), EAAT4 (refs 9,10,11) and EAAT5 (ref. 5) are neuronal. Studies of EAAT4 and EAAC1 indicate an extrasynaptic localization on perisynaptic membranes that are near release sites8,9,10. This localization facilitates rapid Glutamate binding, and may have a role in shaping the amplitude of postsynaptic responses in densely packed cerebellar terminals12,13,14,15. We have used a yeast two-hybrid screen to identify interacting proteins that may be involved in regulating EAAT4—the Glutamate Transporter expressed predominately in the cerebellum—or in targeting and/or anchoring or clustering the Transporter to the target site. Here we report the identification and characterization of two proteins, GTRAP41 and GTRAP48 (for Glutamate Transporter EAAT4 associated protein) that specifically interact with the intracellular carboxy-terminal domain of EAAT4 and modulate its Glutamate transport activity.
Niels C. Danbolt - One of the best experts on this subject based on the ideXlab platform.
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sod1 mutants linked to amyotrophic lateral sclerosis selectively inactivate a glial Glutamate Transporter
Nature Neuroscience, 1999Co-Authors: Davide Trotti, Niels C. Danbolt, Andreas Rolfs, Robert H Brown, Matthias A HedigerAbstract:The mechanism by which Cu2+/Zn2+ superoxide dismutase (SOD1) mutants lead to motor neuron degeneration in familial amyotrophic lateral sclerosis (FALS) is unknown. We show that oxidative reactions triggered by hydrogen peroxide and catalyzed by A4V and I113T mutant but not wild-type SOD1 inactivated the Glutamate Transporter human GLT1. Chelation of the copper ion of the prosthetic group of A4V prevented GLT1 inhibition. GLT1 was a selective target of oxidation mediated by SOD1 mutants, and its reactivity was confined to the intracellular carboxyl-terminal domain. The antioxidant Mn(III)TBAP rescued GLT1 from inhibition. Because inactivation of GLT1 results in neuronal degeneration, we propose that toxic properties of SOD1 mutants lead to neuronal death via an excitotoxic mechanism in SOD1-linked FALS.
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hippocampal gaba and Glutamate Transporter immunoreactivity in patients with temporal lobe epilepsy
Neurology, 1999Co-Authors: Gary W Mathern, Niels C. Danbolt, Delia Mendoza, Alana Lozada, James K Pretorius, Yvette Dehnes, Nathan Nelson, Joao Pereira Leite, Leila Chimelli, Donald E BornAbstract:Objective: Sodium-coupled Transporters remove extracellular neurotransmitters and alterations in their function could enhance or suppress synaptic transmission and seizures. This study determined hippocampal gamma-aminobutyric acid (GABA) and Glutamate Transporter immunoreactivity (IR) in temporal lobe epilepsy (TLE) patients. Methods: Hippocampal sclerosis (HS) patients (n = 25) and non-HS cases (mass lesion and cryptogenic; n = 20) were compared with nonseizure autopsies (n = 8). Hippocampal sections were studied for neuron densities along with IR for Glutamate decarboxylase (GAD; presynaptic GABA terminals), GABA Transporter-1 (GAT-1; presynaptic GABA Transporter), GAT-3 (astrocytic GABA Transporter), excitatory amino acid Transporter 3 (EAAT3; postsynaptic Glutamate Transporter), and EAAT2-1 (glial Glutamate Transporters). Results: Compared with autopsies, non-HS cases with similar neuron counts showed: 1) increased GAD IR gray values (GV) in the fascia dentata outer molecular layer (OML), hilus, and stratum radiatum; 2) increased GAT-1 OML GVs; 3) increased astrocytic GAT-3 GVs in the hilus and Ammon’s horn; and 4) no IR differences for EAAT3-1. HS patients with decreased neuron densities demonstrated: 1) increased OML and inner molecular layer GAD puncta; 2) decreased GAT-1 puncta relative to GAD in the stratum granulosum and pyramidale; 3) increased GAT-1 OML GVs; 4) decreased GAT-3 GVs; 5) increased EAAT3 IR on remaining granule cells and pyramids; 6) decreased glial EAAT2 GVs in the hilus and CA1 stratum radiatum associated with neuron loss; and 7) increased glial EAAT1 GVs in CA2/3 stratum radiatum. Conclusions: Hippocampal GABA and Glutamate Transporter IR differ in TLE patients compared with autopsies. These data support the hypothesis that excitatory and inhibitory neurotransmission and seizure susceptibility could be altered by neuronal and glial Transporters in TLE patients.
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Localization of the Glutamate Transporter protein GLAST in rat retina
Brain research, 1997Co-Authors: Knut P. Lehre, Svend Davanger, Niels C. DanboltAbstract:Glutamate is a neurotransmitter in retina. Glutamate Transporter proteins keep the resting extracellular Glutamate concentration low. This is required for normal neurotransmission and prevents the extracellular concentration of Glutamate from reaching toxic levels. Here we describe the light and electron microscopic localization of the Glutamate Transporter protein GLAST in rat retina using an antibody raised and affinity purified against a peptide corresponding to amino acid residues 522-541. The strongest immunocytochemical labelling was observed in the outer plexiform layer, ganglion cell layer, and optic disc. GLAST was found in Muller cell processes in all retinal layers, notably ensheathing the photoreceptor terminals in the outer plexiform layer, and in astrocytes close to vessels in the inner retina and optic disc. No labelling was observed in neurons. The electrophoretic mobility of GLAST in retina was similar to that in cerebellum. In conclusion, the findings are in agreement with those reported by Derouiche and Rauen [7], except that we did not detect any GLAST in the retinal pigment epithelium.
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Glutamate receptor agonists up-regulate Glutamate Transporter GLAST in astrocytes
Neuroreport, 1996Co-Authors: Georgi Gegelashvili, Niels C. Danbolt, Gianluca Civenni, Giorgio Racagni, Inger Schousboe, Arne SchousboeAbstract:LONG-TERM treatment of astrocytes in primary culture with L-Glutamate (0.1-3 mM) resulted in a dose-dependent increase in D-[ 3 H]aspartate uptake. The effect was abolished by an antagonist of kainate/AMPA receptors, CNQX, and mimicked by kainate, but not by AMPA or tACPD. Both Glutamate and kainate caused a dramatic up-regulation (82% and 69%, respectively) of GLAST, a predominant Glutamate Transporter in cultured astroglia, though the mRNA levels appeared unaffected. Long-term treatment of cultures with dBcAMP stimulated D-[ 3 H]aspartate uptake as well as GLAST expression. Apart from Glutamate, none of the agonists used was capable of increasing further the uptake capacity of the dBcAMP-treated astroglia. The Glutamate receptor-dependent modulation of Glutamate transport in astro-glial cultures may represent a novel feedback regulatory mechanism for Glutamate uptake in the brain.
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Brain Glutamate Transporter Proteins Form Homomultimers
The Journal of biological chemistry, 1996Co-Authors: Øyvind Haugeto, Knut P. Lehre, Kyrre Ullensvang, Line M. Levy, Farrukh A. Chaudhry, Tage Honore, Mogens M. Nielsen, Niels C. DanboltAbstract:Abstract Removal of excitatory amino acids from the extracellular fluid is essential for synaptic transmission and for avoiding excitotoxicity. The removal is accomplished by Glutamate Transporters located in the plasma membranes of both neurons and astroglia. The uptake system consists of several different Transporter proteins that are carefully regulated, indicating more refined functions than simple transmitter inactivation. Here we show by chemical cross-linking, followed by electrophoresis and immunoblotting, that three rat brain Glutamate Transporter proteins (GLAST, GLT and EAAC) form homomultimers. The multimers exist not only in intact brain membranes but also after solubilization and after reconstitution in liposomes. Increasing the cross-linker concentration increased the immunoreactivity of the bands corresponding to trimers at the expense of the dimer and monomer bands. However, the immunoreactivities of the dimer bands did not disappear, indicating a mixture of dimers and trimers. GLT and GLAST do not complex with each other, but as demonstrated by double labeling post-embedding electron microscopic immunocytochemistry, they co-exist side by side in the same astrocytic cell membranes. The oligomers are held together noncovalently in vivo. In vitro, oxidation induces formation of covalent bonds (presumably -S-S-) between the subunits of the oligomers leading to the appearance of oligomer bands on SDS-polyacrylamide gel electrophoresis. Immunoprecipitation experiments suggest that GLT is the quantitatively dominant Glutamate Transporter in the brain. Radiation inactivation analysis gives a molecular target size of the functional complex corresponding to oligomeric structure. We postulate that the Glutamate Transporters operate as homomultimeric complexes.
Michael P Kavanaugh - One of the best experts on this subject based on the ideXlab platform.
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Glutamate Transporter control of ambient Glutamate levels.
Neurochemistry International, 2014Co-Authors: Denis Shchepakin, Leonid V. Kalachev, Michael P KavanaughAbstract:Abstract Accurate knowledge of the ambient extracellular Glutamate concentration in brain is required for understanding its potential impacts on tonic and phasic receptor signaling. Estimates of ambient Glutamate based on microdialysis measurements are generally in the range of ∼2–10 μM, approximately 100-fold higher than estimates based on electrophysiological measurements of tonic NMDA receptor activity (∼25–90 nM). The latter estimates are closer to the low nanomolar estimated thermodynamic limit of Glutamate Transporters. The reasons for this discrepancy are not known, but it has been suggested that microdialysis measurements could overestimate ambient extracellular Glutamate because of reduced Glutamate Transporter activity in a region of metabolically impaired neuropil adjacent to the dialysis probe. We explored this issue by measuring diffusion gradients created by varying membrane densities of Glutamate Transporters expressed in Xenopus oocytes. With free diffusion from a pseudo-infinite 10 μM Glutamate source, the surface concentration of Glutamate depended on Transporter density and was reduced over 2 orders of magnitude by Transporters expressed at membrane densities similar to those previously reported in hippocampus. We created a diffusion model to simulate the effect of transport impairment on microdialysis measurements with boundary conditions corresponding to a 100 μm radius probe. A gradient of metabolic disruption in a thin (∼100 μm) region of neuropil adjacent to the probe increased predicted [Glu] in the dialysate over 100-fold. The results provide support for electrophysiological estimates of submicromolar ambient extracellular [Glu] in brain and provide a possible explanation for the higher values reported using microdialysis approaches.
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macroscopic and microscopic properties of a cloned Glutamate Transporter chloride channel
The Journal of Neuroscience, 1998Co-Authors: Jacques I Wadiche, Michael P KavanaughAbstract:The behavior of a Cl− channel associated with a Glutamate Transporter was studied using intracellular and patch recording techniques in Xenopus oocytes injected with human EAAT1 cRNA. Channels could be activated by application of Glutamate to either face of excised membrane patches. The channel exhibited strong selectivity for amphipathic anions and had a minimum pore diameter of ∼5A. Glutamate flux exhibited a much greater temperature dependence than Cl− flux. Stationary and nonstationary noise analysis was consistent with a sub-femtosiemen Cl− conductance and a maximum channel P o ≪ 1. The Glutamate binding rate was similar to estimates for receptor binding. After Glutamate binding, channels activated rapidly followed by a relaxation phase. Differences in the macroscopic kinetics of channels activated by concentration jumps of l-Glutamate or d-aspartate were correlated with differences in uptake kinetics, indicating a close correspondence of channel gating to state transitions in the Transporter cycle.
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macroscopic and microscopic properties of a cloned Glutamate Transporter chloride channel
The Journal of Neuroscience, 1998Co-Authors: Jacques I Wadiche, Michael P KavanaughAbstract:The behavior of a Cl- channel associated with a Glutamate Transporter was studied using intracellular and patch recording techniques in Xenopus oocytes injected with human EAAT1 cRNA. Channels could be activated by application of Glutamate to either face of excised membrane patches. The channel exhibited strong selectivity for amphipathic anions and had a minimum pore diameter of approximately 5A. Glutamate flux exhibited a much greater temperature dependence than Cl- flux. Stationary and nonstationary noise analysis was consistent with a sub-femtosiemen Cl- conductance and a maximum channel Po << 1. The Glutamate binding rate was similar to estimates for receptor binding. After Glutamate binding, channels activated rapidly followed by a relaxation phase. Differences in the macroscopic kinetics of channels activated by concentration jumps of L-Glutamate or D-aspartate were correlated with differences in uptake kinetics, indicating a close correspondence of channel gating to state transitions in the Transporter cycle.
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flux coupling in a neuronal Glutamate Transporter
Nature, 1996Co-Authors: Noa Zerangue, Michael P KavanaughAbstract:Synaptic transmission is commonly terminated by diffusion and reuptake of neurotransmitter from the synaptic cleft. Glutamate reuptake prevents neurotoxicity and sets the lower limit for the concentration of extracellular Glutamate, so it is important to understand the thermodynamics of this process. Here we use voltage clamping with a pH-sensitive fluorescent dye to monitor electrical currents and pH changes associated with flux of Glutamate mediated by the human neuronal Glutamate Transporter EAAT3. In contrast to a previous model, we find that three sodium ions and one proton are cotransported with each Glutamate ion into the cell, while one potassium ion is transported out of the cell. This coupling can support a transmembrane Glutamate concentration gradient ([Glu]in/[Glu]out) exceeding 10(6) under equilibrium conditions, and would allow the Transporter to continue removing Glutamate over a wide range of ionic conditions.
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kinetics of a human Glutamate Transporter
Neuron, 1995Co-Authors: Jacques I Wadiche, Jeffrey L Arriza, Susan G Amara, Michael P KavanaughAbstract:Currents mediated by a Glutamate Transporter cloned from human motor cortex were measured in Xenopus oocytes. In the absence of Glutamate, voltage jumps induced Na(+)-dependent capacitive currents that were blocked by kainate, a competitive transport antagonist. The pre-steady-state currents can be described by an ordered binding model in which a voltage-dependent Na+ binding is followed by a voltage-independent kainate binding. At -80 mV, two charges are translocated per molecule of Glutamate, with a cycling time of approximately 70 ms, which is significantly slower than the predicted time course of synaptically released Glutamate. The results suggest that Glutamate diffusion and binding to Transporters, rather than uptake, are likely to dominate the synaptic concentration decay kinetics.