Rat Glutamate Transporter Research Articles

Pre-Steady-State Kinetics and Reverse Transport in Rat Glutamate Transporter EAAC1 with an Immobilized Transport Domain

Plasma membrane glutamate transporters move glutamate across the cell membrane in a process that is thought to involve elevator-like movement of the transport domain relative to the static trimerization domain. Conformational changes associated with this elevator-like movement have been blocked by covalent crosslinking of cysteine pairs inserted strategically in several positions in the transporter structure, resulting in inhibition of steady-state transport activity. However, it is not known how these crosslinking restraints affect other partial reactions of the transporter that were identified based on pre-steady-state kinetic analysis. Here, we re-examine two different introduced cysteine pairs in the rat glutamate transporter EAAC1 recombinantely expressed in HEK293 cells, W440C/K268C and K64C/V419C, with respect to the molecular mechanism of their impairment of transporter function.Pre-steady-state kinetic studies of glutamate-induced partial reactions were performed using laser photolysis of caged glutamate to achieve sub-millisecond time resolution. Crosslinking of both cysteine pairs abolished steady-state transport current, as well as the majority of pre-steady-state glutamate-induced charge movements, in both forward and reverse transport mode, suggesting that it is not only the elevator-like movement associated with translocation, but also other transporter partial reactions that are inhibited. In contrast, sodium binding to the empty transporter, and glutamate-induced anion conductance were still intact after the W440C/K268C crosslink. Our results add to the previous mechanistic view of how covalent restraints of the transporter affect function and structural changes linked to individual steps in the transport cycle.

Effects of clonidine on the activity of the rat glutamate transporter EAAT3 expressed inXenopusoocytes

BackgroundClonidine has been shown to be a potent neuroprotectant by acting at α2 receptors on glutamatergic neurons to inhibit the release of glutamate. The aim of this study is to investigate the effects of clonidine on the activity of EAAT3 that can regulate extracellular glutamate.MethodsEAAT3 was expressed in the Xenopus oocytes. Using a two-electrode voltage clamp, membrane currents were recorded after application of 30 µM L-glutamate both in the presence and absence of various concentrations of clonidine. To determine the effects of clonidine on the Km and Vmax of EAAT3 and the reversibility of clonidine effects, membrane currents were recorded after the application of various concentrations of L-glutamate both in the presence and absence of 1.50 × 10-7 M clonidine.ResultsClonidine reduced the EAAT3 responses to L-glutamate in a concentration-dependent manner. This inhibition was statistically significant at higher concentrations than at the clinically relevant range. Clonidine at 1.50 × 10-7 M reduced the Vmax, but did not affect the Km of EAAT3 for L-glutamate.ConclusionsThese results suggest that the direct inhibition of EAAT3 activity is not related to the sedation effect of clonidine and that the clonidine-induced reduction of EAAT3 activity provides additional data for the possible involvement of glutamatergic hyperactivity in the proconvulsant effect of clonidine.

Corticosterone decreases the activity of rat glutamate transporter type 3 expressed in Xenopus oocytes

Glucocorticoids can increase the extracellular concentrations of glutamate, the major excitatory neurotransmitter. We investigated the effects of corticosterone on the activity of a glutamate transporter, excitatory amino acid carrier 1 (EAAC1; also called excitatory amino acid transporter type 3 [EAAT3]), and the roles of protein kinase C (PKC) and phosphatidylinositol 3-kinase (PI3K) in regulating these effects. Rat EAAC1 was expressed in Xenopus oocytes by injecting mRNA. l-Glutamate (30 μM)-induced membrane currents were measured using the two-electrode voltage clamp technique. Exposure of these oocytes to corticosterone (0.01–1 μM) for 72 h decreased EAAC1 activity in a dose-dependent fashion, and this inhibition was incubation time-dependent. Corticosterone (0.01 μM for 72 h) significantly decreased the V max, but not the K m, of EAAC1 for glutamate. Furthermore, pretreatment of oocytes with staurosporine, a PKC inhibitor, significantly decreased EAAC1 activity (1.00 ± 0.06 to 0.70 ± 0.05 μC; P < 0.05). However, no statistical differences were observed between oocytes treated with staurosporine, corticosterone, or corticosterone plus staurosporine. Similar patterns of responses were achieved by chelerythrine or calphostin C, other PKC inhibitors. Phorbol-12-myristate-13-acetate (PMA), a PKC activator, inhibited corticosterone-induced reduction in EAAC1 activity. Pretreating oocytes with wortmannin or LY294002, PI3K inhibitors, also significantly reduced EAAC1 activity, but no difference was observed between oocytes treated with wortmannin, corticosterone, or wortmannin plus corticosterone. The above results suggest that corticosterone exposure reduces EAAC1 activity and this effect is PKC- and PI3K-dependent.

Amitriptyline inhibits the activity of the rat glutamate transporter EAAT3 expressed in Xenopus oocytes

Abstract Objectives Evidence suggests that glutamatergic systems may be involved in the pathophysiology of major depression and the mechanism of action of antidepressants. We have investigated the effects of amitriptyline, a tricyclic antidepressant, on the activity of the excitatory amino acid transporter type 3 (EAAT3), a protein that can regulate extracellular glutamate concentrations in the brain. Methods EAAT3 was expressed in Xenopus oocytes. Using a two-electrode voltage clamp, membrane currents were recorded after application of 30 μM l-glutamate in the presence or absence of various concentrations of amitriptyline or after application of various concentrations of l-glutamate in the presence or absence of 0.64 μM amitriptyline. Key findings Amitriptyline concentration-dependently reduced EAAT3 activity. This inhibition reached statistical significance at 0.38–1.27 μM amitriptyline. Amitriptyline 0.64 μM reduced the pharmacokinetic parameter Vmax, but did not affect the pharmacokinetic parameter Km, of EAAT3 for l-glutamate. The amitriptyline inhibition disappeared after a 4-min washout. Phorbol-12-myristate-13-acetate, a protein kinase C activator, increased EAAT3 activity. However, 0.64 μM amitriptyline induced a similar degree of decrease in EAAT3 activity in the presence or absence of phorbol-12-myristate-13-acetate. Conclusions Our results suggested that amitriptyline at clinically relevant concentrations reversibly reduced EAAT3 activity via decreasing its maximal velocity of glutamate transporting function. The effects of amitriptyline on EAAT3 activity may have represented a novel site of action for amitriptyline to increase glutamatergic neuro-transmission. Protein kinase C may not have been involved in the effects of amitriptyline on EAAT3.

Amitriptyline inhibits the activity of the rat glutamate transporter EAAT3 expressed in &lt;I&gt;Xenopus&lt;/I&gt; oocytes

Evidence suggests that glutamatergic systems may be involved in the pathophysiology of major depression and the mechanism of action of antidepressants. We have investigated the effects of amitriptyline, a tricyclic antidepressant, on the activity of the excitatory amino acid transporter type 3 (EAAT3), a protein that can regulate extracellular glutamate concentrations in the brain. EAAT3 was expressed in Xenopus oocytes. Using a two-electrode voltage clamp, membrane currents were recorded after application of 30 microM L-glutamate in the presence or absence of various concentrations of amitriptyline or after application of various concentrations of L-glutamate in the presence or absence of 0.64 microM amitriptyline. Amitriptyline concentration-dependently reduced EAAT3 activity. This inhibition reached statistical significance at 0.38-1.27 microM amitriptyline. Amitriptyline 0.64 microM reduced the pharmacokinetic parameter Vmax, but did not affect the pharmacokinetic parameter Km, of EAAT3 for L-glutamate. The amitriptyline inhibition disappeared after a 4-min washout. Phorbol-12-myristate-13-acetate, a protein kinase C activator, increased EAAT3 activity. However, 0.64 microM amitriptyline induced a similar degree of decrease in EAAT3 activity in the presence or absence of phorbol-12-myristate-13-acetate. Our results suggested that amitriptyline at clinically relevant concentrations reversibly reduced EAAT3 activity via decreasing its maximal velocity of glutamate transporting function. The effects of amitriptyline on EAAT3 activity may have represented a novel site of action for amitriptyline to increase glutamatergic neurotransmission. Protein kinase C may not have been involved in the effects of amitriptyline on EAAT3.

Critical Role of S465 in Protein Kinase C-Increased Rat Glutamate Transporter Type 3 Activity

Glutamate transporters, also called excitatory amino acid transporters (EAATs), uptake extracellular glutamate and regulate neurotransmission. Activation of protein kinase C (PKC) increases the activity of EAAT type 3 (EAAT3), the major neuronal EAAT. We designed this study to determine which amino acid residue(s) in EAAT3 may be involved in this PKC effect. Selective potential PKC phosphorylation sites were mutated. These EAAT3 mutants were expressed in the Xenopus oocytes. Phorbol 12-myristate 13-acetate, a PKC activator, significantly increased wild-type EAAT3 activity. Mutation of serine 465 to alanine or aspartic acid, but not the mutation of threonine 5 to alanine, abolished PKC-increased EAAT3 activity. Our results suggest a critical role of serine 465 in the increased EAAT3 activity by PKC activation.

Critical role of S465 in the increased rat glutamate transporter type 3 activity after protein kinase C activation

Critical role of S465 in the increased rat glutamate transporter type 3 activity after protein kinase C activation

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

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 (approximately 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 approximately 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 [(3)H]-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 microM). Intracellular injection of BAPTA, a Ca(2+)-chelating agent, did not affect the PMA-enhanced Cl(-) currents. 4alpha-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.

Dexamethasone modulates the development of morphine tolerance and expression of glutamate transporters in rats

We recently demonstrated an increase in spinal cerebrospinal fluid (CSF) excitatory amino acids (EAAs) in morphine-tolerant rats after morphine challenge. The present study examined whether co-infusion of the glucocorticoid dexamethasone (DEX) co-infusion inhibited morphine tolerance and the morphine challenge-induced EAAs increase after long-term morphine infusion. Intrathecal (i.t.) catheters and one microdialysis probe were implanted to male Wistar rats. Rats were divided into four groups: i.t. morphine (15μg/h), saline (1μl/h), DEX (2μg/h), or DEX (2μg/h) plus morphine (15μg/h) infusion for 5 days. Tail-flick responses were examined before drug infusion and daily after the start of infusion for 5 days. Moreover, on day 5 after morphine challenge (50μg, i.t.), CSF EAAs was also measured. Rat spinal cords were removed on day 5, and prepared for Western blot analysis of different glutamate transporters (GTs). The AD 50 (analgesic dose) on day 5 was 1.33μg in saline-infused rats, 83.84μg in morphine-tolerant rats, and 10.15μg in DEX plus morphine co-infused rats. Single DEX (2μg, i.t.) injection did not enhance morphine’s antinociceptive effect in either naïve or morphine-tolerant rats. No difference in CSF EAA level was observed in all groups between baseline (before drug infusion) and on day 5 after tolerance developed. Surprisingly, on day 5, after morphine challenge, an increase in glutamate and aspartate (284±47% and 201±18% of basal) concentration was observed, and morphine lost its antinociceptive effect (maximum percent effect, MPE=41±12%), whereas DEX/morphine co-infusion inhibited morphine-evoked EAA increase with a MPE=97±2%. DEX co-infusion prevented the downregulation of glial glutamate transporters (GLAST (Glu-Asp transporter) and GLT-1 (Glu transporter-1)), but not the neuronal GT EAAC1 (excitatory amino acid carrier). Upregulation of GLT-1 was also observed (204±20% of basal). DEX co-infusion inhibits the morphine-challenge induced EAA increase and prevents the loss of morphine’s antinociceptive effect after long-term morphine infusion.

Effects of propofol on the activity of rat glutamate transporter type 3 expressed in Xenopus oocytes: the role of protein kinase C

We investigated the effects of propofol on one type of glutamate transporter, excitatory amino acid transporter 3 (EAAT3) and the role of protein kinase C (PKC) in mediating these effects. Rat EAAT3 was expressed in Xenopus oocytes. l-glutamate (30 μM)-induced membrane currents were measured. Propofol increased glutamate-induced inward currents significantly at two tested concentrations (30 and 100 μM) but not at other concentrations. Propofol (30 μM) significantly increased V max, but not K m of EAAT3 for glutamate. The combination of phorbol-12-myrisate-13-acetate (PMA, a PKC activator) and propofol did not increase the responses further compared with PMA or propofol alone. Three PKC inhibitors (staurosporine, calphostin C, and chelerythrine) did not affect basal EAAT3 activity but significantly inhibited the propofol-enhanced EAAT3 activity. Our results suggest that propofol enhances EAAT3 activity at clinically relevant concentrations and PKC may mediate these effects.

Chronic exposure of nicotine modulates the expressions of the cerebellar glial glutamate transporters in rats.

Rats were given nicotine (25 ppm) in their drinking water at the start of their mating period in order to study the expressions of glutamate transporter subtypes in cerebellar astrocytes following the chronic exposure of nicotine after mating. After the offspring were delivered, each group was divided into two subgroups. One group, the control group, was given distilled water only and the other group, the experimental group, was given distilled water containing nicotine. The cerebellar astrocytes were prepared from 7 day-old pups at each group. Ten days after the cells were cultured, the expression of the glutamate transporter subtypes (GLAST and GLT-1) was determined using immunochemistry and immunoblotting. During the continuous treatments, the developmental expression patterns of the GLAST and GLT-1 in the cerebellum were also determined from 2, 4 and 8 week-old rats. The expression levels of GLAST in cultured astrocytes of both the pre- or post-natally exposed groups were higher than those of the control group. However, these expression levels of the continuously exposed group were lower than those of the control group. Compared to those of the control group, the GLT-1 expression levels of all the nicotine-treated groups were higher, particularly in the continuously treated group. According to the results from the immochemistry procedure, the cerebellar GLAST and GLT-1 expression levels of all nicotine-treated groups were lower than those of the control group at each age. However, the immunoblotting procedure showed that the cerebellar GLT-1 expression levels of all the nicotine-treated groups were higher than those of the control group, except for the rats that were continuously exposed for 8 weeks using immunoblotting. These results suggest that the expression of the glial GLAST and GLT-1 are altered differently depending on the initial exposure time and the particular period of nicotine exposure. In addition, nicotine exposure during gestation has persistent effects on glial cells.

The different responses of rat glutamate transporter type 2 and its mutant (tyrosine 403 to histidine) activity to volatile anesthetics and activation of protein kinase C

Glutamate transporters play an important role in homeostasis of extracellular glutamate, a major excitatory neurotransmitter and a potential neurotoxin. In mammalian brain, glutamate transporter type 2 (EAAT2) is the most abundant form. Studies of molecular structures demonstrated that tyrosine 403 is critical in regulating the ion selectivity and transport mode of EAAT2. We hypothesized that wild type EAAT2 and its mutant at tyrosine 403 have different responses to volatile anesthetics, commonly used anesthetics that have been shown to affect glutamate transporter activity and decrease extracellular glutamate concentrations. We used site-directed mutagenesis and oocyte expression systems to test the hypothesis. Volatile anesthetics did not affect the activity of wild type EAAT2, isolated from rat hippocampus. When tyrosine 403 was replaced by histidine (Y403H), volatile anesthetics (isoflurane or halothane) at clinically relevant concentrations significantly decreased the transporter activity. Okadaic acid, a phosphatase inhibitor, significantly prolonged the isoflurane-induced inhibition. This inhibition was reversed by staurosporine and calphostin C, two protein kinase C (PKC) inhibitors, but not by the third PKC inhibitor, chelerythrine. Phorbol 12-myristate 13-acetate, a PKC activator, inhibited the activity of both wild type and Y403H EAAT2. This inhibition was also reversed by the same two PKC inhibitors but not by the third one. These results suggest that the switch of tyrosine 403 to histidine rendered EAAT2 sensitive to volatile anesthetics, a phenomenon that may require protein phosphorylation. PKC may be involved in the regulation of the activity of both wild type and Y403H EAAT2.

Decreased expression of glutamate transporters in genetic absence epilepsy rats before seizure occurrence.

In absence epilepsy, epileptogenic processes are suspected of involving an imbalance between GABAergic inhibition and glutamatergic excitation. Here, we describe alteration of the expression of glutamate transporters in rats with genetic absence (the Genetic Absence Epilepsy Rats from Strasbourg: GAERS). In these rats, epileptic discharges, recorded in the thalamo-cortical network, appear around 40 days after birth. In adult rats no alteration of the protein expression of the glutamate transporters was observed. In 30-day-old GAERS protein levels (quantified by western blot) were lower in the cortex by 21% and 35% for the glial transporters GLT1 and GLAST, respectively, and by 32% for the neuronal transporter EAAC1 in the thalamus compared to control rats. In addition, the expression and activity of GLAST were decreased by 50% in newborn GAERS cortical astrocytes grown in primary culture. The lack of modification of the protein levels of glutamatergic transporters in adult epileptic GAERS, in spite of mRNA variations (quantified by RT-PCR), suggests that they are not involved in the pathogeny of spike-and-wave discharges. In contrast, the alteration of glutamate transporter expression, observed before the establishment of epileptic discharges, could reflect an abnormal maturation of the glutamatergic neurone-glia circuitry.

LY341495 is a nanomolar potent and selective antagonist of group II metabotropic glutamate receptors

The in vitro pharmacology of a structurally novel compound, LY341495, was investigated at human recombinant metabotropic glutamate (mGlu) receptor subtypes expressed in non-neuronal (RGT, rat glutamate transporter) cells. LY341495 was a nanomolar potent antagonist of 1 S,3 R-1-aminocyclopentane-1,3-dicarboxylic acid (ACPD)-induced inhibition of forskolin-stimulated cAMP formation at mGlu2 and mGlu3 receptors (respective IC 50s of 0.021 and 0.014 μM). At group I mGlu receptor expressing cells, LY341495 was micromolar potent in antagonizing quisqualate-induced phosphoinositide (PI) hydrolysis, with IC 50 values of 7.8 and 8.2 μM for mGlu1a and mGlu5a receptors, respectively. Among the human group III mGlu receptors, the most potent inhibition of l-2-amino-4-phosphonobutyric acid ( l-AP4) responses was seen for LY341495 at mGlu8, with an IC 50 of 0.17 μM. LY341495 was less potent at mGlu7 (IC 50=0.99 μM) and least potent at mGlu4 (IC 50=22 μM). Binding studies in rat brain membranes also demonstrated nanomolar potent group II mGlu receptor affinity for LY341495, with no appreciable displacement of ionotropic glutamate receptor ligand binding. Thus, LY341495 has a unique range of selectivity across the mGlu receptor subtypes with a potency order of mGlu3≥mGlu2>mGlu8>mGlu7⪢mGlu1a=mGlu5a>mGlu4. In particular, LY341495 is the most potent antagonist yet reported at mGlu2, 3 and 8 receptors. Thus, it represents a novel pharmacological agent for elucidating the function of mGlu receptors in experimental systems.

Neuronal and Glial Glutamate Transporters Possess an SH‐based Redox Regulatory Mechanism

Glutamate uptake into nerve cells and astrocytes via high-affinity transporters controls the extracellular glutamate concentration in the brain, with major implications for physiological excitatory neurotransmission and the prevention of excitotoxicity. We report here that three recently cloned rat glutamate transporter subtypes, viz. EAAC1 (neuronal), GLT1 and GLAST (glial), possess a redox-sensing property, undergoing opposite functional changes in response to oxidation or reduction of reactive sulphydryls present in their structure. In particular, thiol oxidation with 5,5'-dithio-bis(2-nitrobenzoic) acid (DTNB) and disulphide reduction with dithiothreitol (DTT) result, respectively, in reduced and increased uptake capacity by a preparation of partially purified brain transporters as well as by the three recombinant proteins reconstituted into liposomes. In this model system, EAAC1, GLT1 and GLAST react similarly to DTT/DTNB exposures despite their different contents of cysteines, suggesting that only the conserved residues might be involved in redox modulation. Redox sensitivity is a property of the glutamate transporters also when present in their native cell environment. Thus, by using cultured cortical astrocytes and the whole-cell patch-clamp technique we were able to observe dynamic increase and decrease of the glutamate uptake current in response to application of DTT and DTNB in sequence. Moreover, in the same paradigm, DDT-reversible current inhibition was observed with hydrogen peroxide instead of DTNB, indicating that the SH-based redox modulatory site is targeted by endogenous oxidants and might constitute an important physiological or pathophysiological regulatory mechanism of glutamate uptake in vivo.

Abolition of substrate-dependent currents by tyrosine mutation in the transmembrane domain of glutamate transporter

By site-directed mutagenesis we examined the roles of tyrosine residues (Tyr127) in the putative transmembrane domain of rat glutamate transporter (GLAST). When expressed in Xenopus oocytes, Y127F mutant protein, which was localized in plasma membranes of oocytes, completely abolished glutamate uptake currents but did not affect the intrinsic substrate-independent currents. Coexpression of wild type and mutant transporters supports that the Y127F mutation did not elicit glutamate efflux. The efflux of glutamate by wild type or Y127F mutant transporters was measured under the condition of ion perturbation where transporters run in the reverse direction.©1997 Federation of European Biochemical Societies.

Pharmacological characterization of a cloned rat glutamate transporter (GluT-1)

Pharmacological properties of a cDNA clone encoding a high affinity, Na +-dependent l-glutamate transporter (GluT-1) were examined using Xenopus oocytes. l-[ 3H]glutamate transport was inhibited by the putative endogenous substrates l-aspartate ( K i = 65 μM) and l-glutamate ( K i = 70 μM). l-Homocysteate did not significantly inhibit high-affinity glutamate transport ( K i = 2.7 mM). Of the previously identified uptake inhibitors, DL-threo-β-hydroxyaspartate ( K i = 65 μM), l-cysteine sulfinate ( K i = 80 μM), β-glutamate ( K i = 475 μM) and l-aspartate-β-hydroxamate ( K i = 950 μM) inhibited l-glutamate uptake. The other l-glutamate uptake blockers examined, including dihydrokainate, l-α-aminoadipate and SITS, weakly inhibited uptake of l-glutamate with K i values in excess of 1 mM. These features of the inhibitor sensitivities of GluT-1 transport show that GluT-1 is less sensitive to these agents than previously characterized glutamate transporters in rat brain, suggesting that GluT-1 is a novel glutamate transporter with a unique pharmacologic profile.

Expression cloning of a rat glutamate transporter

A functional cDNA clone for the rat glutamate transporter was isolated by a cloning approach using a Xenopus oocyte expression system. The cDNA sequence predicts a protein of 543 amino acids with 6 putative transmembrane domains. The glutamate transporter shows no sequence similarity to other members of the known neurotransmitter transporter family. Expression of the cDNA clone in Xenopus oocyte indicates that glutamate transport activity is Na +- and Cl −-dependent and sensitive to selective glutamate transporter inhibitor. The glutamate transporter mRNA is expressed in both the brain and the peripheral tissues at different levels.