The role of diurnal fluctuations in excitatory amino acid carrier 1 levels in post-ischemic hippocampal Zn2+ accumulation.

Accumulating evidence indicates time-of-day variations in ischemic neuronal injury. Under ischemic conditions, Zn2+ is massively released from hippocampal glutamatergic neurons, and intracellular Zn2+ accumulation results in neuron death. Notably, excitatory amino acid carrier 1 (EAAC1), known as a cysteine transporter, is involved in Zn2+ homeostasis, and its expressions exhibit a diurnal fluctuation. This study aimed to investigate whether time of day of an ischemic insult affects Zn2+ accumulation and neuronal injury and determine whether altered Zn2+ accumulation is modulated by EAAC1 diurnal fluctuation in the hippocampus in a mouse model of ischemic stroke. Mice subjected to transient global ischemia for 40 min at Zeitgeber time 18 (ZT18) (23:00) exhibited reduced Zn2+ accumulation and neuronal death in the hilar region of the hippocampus compared to those at ZT4 (09:00). The EAAC1 protein expression in the hippocampus was increased at ZT18 relative to ZT4. Intracerebroventricular injection of a non-selective excitatory amino acid transporter inhibitor, DL-threo-ß-benzyloxyaspartate, or a selective EAAC1 inhibitor, L-aspartic acid ß-hydroxamate, increased ischemia-induced Zn2+ accumulation and neuronal death in the hilus at ZT18. These findings suggest that ischemia-induced Zn2+ accumulation displays circadian fluctuations through diurnal variations in EAAC1 expressions and affects susceptibility to ischemic neuronal injury in the hippocampal hilar region.

Identification and Structure-Activity Relationship Study of Imidazo[1,2-a]pyridine-3-amines as First Selective Inhibitors of Excitatory Amino Acid Transporter Subtype 3 (EAAT3).

In the present study, screening of a library of 49,087 compounds at the excitatory amino acid transporter subtype 3 (EAAT3) led to the identification of 2-(furan-2-yl)-8-methyl-N-(o-tolyl)imidazo[1,2-a]pyridin-3-amine (3a) which showed a >20-fold preference for inhibition of EAAT3 (IC50 = 13 µM) over EAAT1,2,4 (EAAT1: IC50 ∼ 250 µM; EAAT2,4: IC50 > 250 µM). It was shown that a small lipophilic substituent (methyl or bromine) at the 7- and/or 8-position was essential for activity. Furthermore, the substitution pattern of the o-tolyl group (compound 5b) and the chemical nature of the substituent in the 2-position (compound 7b) were shown to be essential for the selectivity toward EAAT3 over EAAT1,2. The most prominent analogues to come out of this study are 3a and 3e that display ∼35-fold selectivity for EAAT3 (IC50 = 7.2 µM) over EAAT1,2,4 (IC50 ∼ 250 µM).

Genetically Encoded Halide Sensor-Based Fluorescent Assay for Rapid Screening of Glutamate Transport and Inhibition.

Glutamate is the main excitatory neurotransmitter in the mammalian central nervous system. Excitatory amino acid transporters (EAATs) are a family of transmembrane transporters responsible for glutamate uptake into cells, and their malfunction is related to a variety of diseases, including neurodegenerative diseases and stroke. Screening for and developing inhibitors of EAATs as well as related transporters is a significant field of study for biomedical and pharmaceutical applications. Rapid, high-throughput methods are critical for the study of glutamate transporters, and fluorescent methods are appealing for this purpose as compared to more traditional electrophysiological methods. In this study, we present a method for studying glutamate transporters and inhibitors by utilizing a mutated version of a yellow fluorescent protein (YFP) highly sensitive to quenching by anions (mClY). We applied this YFP variant to fluorescent imaging of anion flux in HEK293 cells caused by transiently expressed excitatory amino acid carrier 1 (EAAC1) and excitatory amino acid transporter 2 (EAAT2) and its inhibition by competitive blockers. This method enables rapid identification of inhibitors and, potentially, activators of EAAT function, which is critical for glutamate transport research.

Chemoenzymatic Synthesis and Pharmacological Characterization of Functionalized Aspartate Analogues As Novel Excitatory Amino Acid Transporter Inhibitors.

Aspartate (Asp) derivatives are privileged compounds for investigating the roles governed by excitatory amino acid transporters (EAATs) in glutamatergic neurotransmission. Here, we report the synthesis of various Asp derivatives with (cyclo)alkyloxy and (hetero)aryloxy substituents at C-3. Their pharmacological properties were characterized at the EAAT1-4 subtypes. The l- threo-3-substituted Asp derivatives 13a-e and 13g-k were nonsubstrate inhibitors, exhibiting pan activity at EAAT1-4 with IC50 values ranging from 0.49 to 15 µM. Comparisons between (dl- threo)-19a-c and (dl- erythro)-19a-c Asp analogues confirmed that the threo configuration is crucial for the EAAT1-4 inhibitory activities. Analogues (3b-e) of l-TFB-TBOA (3a) were shown to be potent EAAT1-4 inhibitors, with IC50 values ranging from 5 to 530 nM. Hybridization of the nonselective EAAT inhibitor l-TBOA with EAAT2-selective inhibitor WAY-213613 or EAAT3-preferring inhibitor NBI-59159 yielded compounds 8 and 9, respectively, which were nonselective EAAT inhibitors displaying considerably lower IC50 values at EAAT1-4 (11-140 nM) than those displayed by the respective parent molecules.

Babesia divergens-infected red blood cells take up glutamate via an EAAT3 independent mechanism.

Human red blood cells infected with the malaria parasite Plasmodium falciparum show an increased permeability to a number of solutes. We have previously demonstrated that such infected cells take up glutamate via a member of the excitatory amino acid transporter protein family (EAAT), namely EAAT3. Babesia divergens is a parasite that also infects human erythrocytes, and also induces increased solute permeability, including for glutamate. Here we have investigated whether glutamate uptake in B. divergens infected human red blood cells is also dependent on EAAT3 activity. We find that, although B. divergens infected cells do take up glutamate, this uptake is independent on EAAT3. Thus, though infecting the same host cell, two related parasites have developed distinct pathways to obtain access to nutrients from the extracellular milieu.

Photoswitchable Inhibitor of a Glutamate Transporter.

Excitatory amino acid transporters clear glutamate from the synaptic cleft and play a critical role in glutamatergic neurotransmission. Their differential roles in astrocytes, microglia, and neurons are poorly understood due in part to a lack of pharmacological tools that can be targeted to specific cells and tissues. We now describe a photoswitchable inhibitor, termed ATT, that interacts with the major mammalian forebrain transporters EAAT1-3 in a manner that can be reversibly switched between trans (high-affinity) and cis (low-affinity) configurations using light of different colors. In the dark, ATT competitively inhibited the predominant glial transporter EAAT2 with ∼200-fold selectivity over the neuronal transporter EAAT3. Brief exposure to 350 nm light reduced the steady-state blocker affinity by more than an order of magnitude. Illumination of EAAT2 complexed with ATT induced a corresponding increase in the blocker off-rate monitored in the presence of glutamate. ATT can be used to reversibly manipulate glutamate transporter activity with light and may be useful to gain insights into the dynamic physiological roles of glutamate transporters in the brain, as well as to study the molecular interactions of transporters with ligands.

ß-Sulfonamido Functionalized Aspartate Analogues as Excitatory Amino Acid Transporter Inhibitors: Distinct Subtype Selectivity Profiles Arising from Subtle Structural Differences.

In this study inspired by previous work on 3-substituted Asp analogues, we designed and synthesized a total of 32 ß-sulfonamide Asp analogues and characterized their pharmacological properties at the excitatory amino acid transporter subtypes EAAT1, EAAT2, and EAAT3. In addition to several potent EAAT inhibitors displaying IC50 values ∼1 µM at all three subtypes, this elaborate structure-activity relationship also identified analogues exhibiting distinct preferences or selectivities for specific transporter subtypes. Introduction of two fluorine atoms on the phenyl ring yielded analogue 4y that displayed an IC50 of 0.8 µM at EAAT1 with a 14- and 9-fold preference over EAAT2 and EAAT3, respectively. Conversely, the m-CF3-phenyl analogue 4r was a potent selective EAAT2-inhibitor (IC50 = 2.8 µM) exhibiting 30- and 50-fold selectivity over EAAT1 and EAAT3, respectively. In conclusion, even small structural differences in these ß-sulfonamide Asp analogues provide analogues with diverse EAAT subtype selectivity profiles.

Concise Asymmetric Synthesis and Pharmacological Characterization of All Stereoisomers of Glutamate Transporter Inhibitor TFB-TBOA and Synthesis of EAAT Photoaffinity Probes.

Glutamate is the major excitatory neurotransmitter in the mammalian brain. Its rapid clearance after the release into the synaptic cleft is vital in order to avoid toxic effects and is ensured by several transmembrane transport proteins, so-called excitatory amino acid transporters (EAATs). Impairment of glutamate removal has been linked to several neurodegenerative diseases and EAATs have therefore received increased attention as therapeutic targets. O-Benzylated l-threo-ß-hydroxyaspartate derivatives have been developed previously as highly potent inhibitors of EAATs with TFB-TBOA ((2S,3S)-2-amino-3-((3-(4-(trifluoromethyl)benzamido)benzyl)oxy)succinic acid) standing out as low-nanomolar inhibitor. We report the stereoselective synthesis of all four stereoisomers of TFB-TBOA in less than a fifth of synthetic steps than the published route. For the first time, the inhibitory activity and isoform selectivity of these TFB-TBOA enantio- and diastereomers were assessed on human glutamate transporters EAAT1-3. Furthermore, we synthesized potent photoaffinity probes based on TFB-TBOA using our novel synthetic strategy.

Intracellular mGluR5 plays a critical role in neuropathic pain.

Spinal mGluR5 is a key mediator of neuroplasticity underlying persistent pain. Although brain mGluR5 is localized on cell surface and intracellular membranes, neither the presence nor physiological role of spinal intracellular mGluR5 is established. Here we show that in spinal dorsal horn neurons >80% of mGluR5 is intracellular, of which ∼60% is located on nuclear membranes, where activation leads to sustained Ca(2+) responses. Nerve injury inducing nociceptive hypersensitivity also increases the expression of nuclear mGluR5 and receptor-mediated phosphorylated-ERK1/2, Arc/Arg3.1 and c-fos. Spinal blockade of intracellular mGluR5 reduces neuropathic pain behaviours and signalling molecules, whereas blockade of cell-surface mGluR5 has little effect. Decreasing intracellular glutamate via blocking EAAT-3, mimics the effects of intracellular mGluR5 antagonism. These findings show a direct link between an intracellular GPCR and behavioural expression in vivo. Blockade of intracellular mGluR5 represents a new strategy for the development of effective therapies for persistent pain.

Doxepin and imipramine but not fluoxetine reduce the activity of the rat glutamate transporter EAAT3 expressed in Xenopus oocytes.

BACKGROUND: Many researchers have suggested that the glutamatergic system may be involved in the effects of antidepressant therapies. We investigated the effects of doxepin, imipramine, and fluoxetine on the excitatory amino acid transporter type 3 (EAAT3). METHODS: EAAT3 was expressed in Xenopus oocytes by injection of EAAT3 mRNA. Membrane currents were recorded after application of L-glutamate (30 µM) in the presence or absence of various concentrations of doxepin, imipramine, and fluoxetine. To study the effects of protein kinase C (PKC) activation on EAAT3 activity, oocytes were pre-incubated with phorbol 12-myristate-13-acetate (PMA) before application of imipramine and doxepin. RESULTS: Doxepin at 0.063-1.58 µM significantly decreased EAAT3 activity. Imipramine reduced EAAT3 activity in a concentration-dependent manner at 0.16-0.95 µM. However, fluoxetine did not affect EAAT3 activity, and PMA increased EAAT3 activity. At 0.32 µM, imipramine caused an equivalent decrease in EAAT3 activity in the presence or absence of PMA. However, 0.79 µM doxepin did not abolish the enhancement of EAAT3 activity by PMA. CONCLUSIONS: We showed that doxepin and imipramine, but not fluoxetine, inhibited EAAT3 activity at clinically relevant concentrations. This reveals a novel mechanism of action for doxepin and imipramine; that they increase glutamatergic neurotransmission. PKC may be involved in the effects of doxepin on EAAT3, but is not involved in the effects of imipramine at the concentrations studied.

Gabapentin inhibits the activity of the rat excitatory glutamate transporter 3 expressed in Xenopus oocytes.

Gabapentin, a derivative of γ-aminobutyric acid (GABA), is used to treat epilepsy and neuropathic pain. The pharmacological mechanisms for gabapentin effects are not completely elucidated. We investigated the effect of gabapentin on the activity of excitatory amino acid transporter 3 (EAAT3) that can regulate extracellular glutamate concentrations. EAAT3 was expressed in Xenopus oocytes. Membrane currents were recorded after application of l-glutamate in the presence or absence of different concentrations of gabapentin (1-300µM) by using a two-electrode voltage clamp. To determine the effect of gabapentin on Vmax and Km of EAAT3 for l-glutamate, l-glutamate at 3-300µM was used. To study the effects of protein kinase C (PKC) and phosphatidylinositol 3-kinase (PI3K) on gabapentin-induced changes in EAAT3 activity, oocytes were incubated with the PKC activator (Phorbol 12-myristate 13-acetate, PMA), the PKC inhibitors (chelerythrine or staurosporine), and the PI3K inhibitor wortmannin. Gabapentin decreased EAAT3 activity in a concentration-dependent manner and EAAT3 activity was significantly inhibited by 10-300µM gabapentin. Gabapentin significantly decreased Vmax without affecting Km. PMA increased EAAT3 activity; however, gabapentin attenuated the PMA-induced increase in EAAT3 activity. Pre-incubation of oocytes with chelerythrine, staurosporine, or wortmannin decreased basal EAAT3 activity, which was further reduced by gabapentin. We conclude that gabapentin decreases EAAT3 activity at clinically relevant and higher concentrations, in which PKC and PI3K may not be involved. The results suggest that EAAT3 might not be a target for the anticonvulsant action of gabapentin.

Ondansetron attenuates the activity of excitatory amino acid transporter type 3 expressed in Xenopus oocytes.

The purpose of this study was to evaluate the effect of ondansetron on excitatory amino acid transporter type 3 (EAAT3) and to elucidate the roles of protein kinase C (PKC) and phosphatidylinositol 3-kinase (PI3K) in the effect. EAAT3 was expressed in Xenopus oocytes following the injection of rat EAAT3 mRNAs. Using the two-electrode voltage clamping method, the inward currents induced by L-glutamate were measured for 1 min in the presence and absence of ondansetron (1-1000 µM). Different concentrations of L-glutamate (3-300 µM) were used to determine the kinetic characteristics of EAAT3. To identify the involvement of PKC and PI3K in the effect, oocytes were exposed to a PKC activator and to PKC inhibitors and PI3K inhibitors, and L-glutamate-induced currents were recorded. Ondansetron decreased EAAT3 activity in a dose-dependent manner. In a kinetic study, ondansetron (10 µM for 3 min) reduced Vmax, but not Km compared with the control group. The PKC activator abolished the ondansetron-induced decrease in EAAT3 activity. The PKC inhibitors (staurosporine and chelerythrine) and ondansetron had not additive or synergistic effects on EAAT3 activity. The PI3K inhibitors (wortmannin and LY294002) decreased the EAAT3 response, although there were no differences among the groups comprising ondansetron, PI3K inhibitors, and PI3K inhibitors plus ondansetron. Our results demonstrate that ondansetron attenuates EAAT3 activity and this effect seems to be mediated by PKC and PI3K.

Morphine induces redox-based changes in global DNA methylation and retrotransposon transcription by inhibition of excitatory amino acid transporter type 3-mediated cysteine uptake.

Canonically, opioids influence cells by binding to a G protein-coupled opioid receptor, initiating intracellular signaling cascades, such as protein kinase, phosphatidylinositol 3-kinase, and extracellular receptor kinase pathways. This results in several downstream effects, including decreased levels of the reduced form of glutathione (GSH) and elevated oxidative stress, as well as epigenetic changes, especially in retrotransposons and heterochromatin, although the mechanism and consequences of these actions are unclear. We characterized the acute and long-term influence of morphine on redox and methylation status (including DNA methylation levels) in cultured neuronal SH-SY5Y cells. Acting via µ-opioid receptors, morphine inhibits excitatory amino acid transporter type 3-mediated cysteine uptake via multiple signaling pathways, involving different G proteins and protein kinases in a temporal manner. Decreased cysteine uptake was associated with decreases in both the redox and methylation status of neuronal cells, as defined by the ratios of GSH to oxidized forms of glutathione and S-adenosylmethionine to S-adenosylhomocysteine levels, respectively. Further, morphine induced global DNA methylation changes, including CpG sites in long interspersed nuclear elements (LINE-1) retrotransposons, resulting in increased LINE-1 mRNA. Together, these findings illuminate the mechanism by which morphine, and potentially other opioids, can influence neuronal-cell redox and methylation status including DNA methylation. Since epigenetic changes are implicated in drug addiction and tolerance phenomenon, this study could potentially extrapolate to elucidate a novel mechanism of action for other drugs of abuse.

Nicotine decreases the activity of glutamate transporter type 3.

Nicotine, the main ingredient of tobacco, elicits seizures in animal models and cigarette smoking is regarded as a behavioral risk factor associated with epilepsy or seizures. In the hippocampus, the origin of nicotine-induced seizures, most glutamate uptake could be performed primarily by excitatory amino acid transporter type 3 (EAAT3). An association between temporal lobe epilepsy and EAAT3 downregulation has been reported. Therefore, we hypothesized that nicotine may elicit seizures through the attenuation of EAAT3 activity. We investigated chronic nicotine exposure (72 h) cause reduction of the activity of EAAT3 in a Xenopus oocyte expression system using a two-electrode voltage clamp. The roles of protein kinase C (PKC) and phosphatidylinositol 3-kinase (PI3K) were also determined. Nicotine (0.001-1 µM) resulted in a time- and dose-dependent decrease in EAAT3 activity with maximal inhibition at nicotine concentrations of 0.03 µM or higher and at an exposure time of 72 h. Vmax on the glutamate response was significantly reduced in the nicotine group (0.03 µM for 72 h), but the Km value of EAAT3 for glutamate was not altered. When nicotine-exposed oocytes (0.03 µM for 72 h) were pretreated with phorbol-12-myristate-13-acetate (PMA, a PKC activator), the nicotine-induced reduction in EAAT3 activity was abolished. PKC inhibitors (staurosporine, chelerythrine, and calphostin C) significantly reduced basal EAAT3 activity, but there were no significant differences among the PKC inhibitors, nicotine, and PKC inhibitors+nicotine groups. Similar response patterns were observed among PI3K inhibitors (wortmannin and LY294002), nicotine, and PI3K inhibitors+nicotine. In conclusion, this study suggests that nicotine decreases EAAT3 activity, and that this inhibition seems to be dependent on PKC and PI3K. Our results may provide an additional mechanism for nicotine-induced seizure.

Riluzole attenuates excitatory amino acid transporter type 3 activity in Xenopus oocytes via protein kinase C inhibition.

This study aimed to evaluate the effect of riluzole on the activity of excitatory amino acid transporter type 3 (EAAT3), a neuronal glutamate transporter, and to investigate the role of protein kinase C (PKC) in this effect. EAAT3 expression was induced in Xenopus oocytes by injecting EAAT3 mRNA. Using the two-electrode voltage clamping method, membrane currents were recorded before, during, and after applying l-glutamate (30 µM) in the absence and presence of prior incubation with riluzole (0.3-100 µM). To study the effect of PKC on the riluzole-induced change in EAAT3 activity, oocytes were preincubated with 100 µM phorbol-12-myristate-13-acetate (PMA), a PKC activator, or PKC inhibitors (2 µM staurosporine and 100 µM chelerythrine) before the recording. Responses were quantified by integrating current traces and are reported in microCoulombs (µC). Riluzole reduced EAAT3 activity in a concentration-dependent manner (0.3-100 µM). Treatment of oocytes with PMA significantly increased the baseline and riluzole-reduced EAAT activity (P<0.05). In addition, treatment of oocytes with PKC inhibitors reduced basal transporter currents, but did not show a further significant decrease in the riluzole-reduced EAAT3 activity. These results suggest that riluzole reduces EAAT3 activity through PKC inhibition.

Functional and morphological characterization of glutamate transporters in the rat locus coeruleus.

BACKGROUND AND PURPOSE: Excitatory amino acid transporters (EAATs) in the CNS contribute to the clearance of glutamate released during neurotransmission. The aim of this study was to explore the role of EAATs in the regulation of locus coeruleus (LC) neurons by glutamate. EXPERIMENTAL APPROACH: We measured the effect of different EAAT subtype inhibitors/enhancers on glutamate- and KCl-induced activation of LC neurons in rat slices. EAAT2-3 expression in the LC was also characterized by immunohistochemistry. KEY RESULTS: The EAAT2-5 inhibitor DL-threo-ß-benzyloxaspartic acid (100 µM), but not the EAAT2, 4, 5 inhibitor L-trans-pyrrolidine-2,4-dicarboxylic acid (100 µM) or the EAAT2 inhibitor dihydrokainic acid (DHK; 100 µM), enhanced the glutamate- and KCl-induced activation of the firing rate of LC neurons. These effects were blocked by ionotropic, but not metabotrobic, glutamate receptor antagonists. DHK (100 µM) was the only EAAT inhibitor that increased the spontaneous firing rate of LC cells, an effect that was due to inhibition of EAAT2 and subsequent AMPA receptor activation. Chronic treatment with ceftriaxone (200 mg·kg(-1) i.p., once daily, 7 days), an EAAT2 expression enhancer, increased the actions of glutamate and DHK, suggesting a functional impact of EAAT2 up-regulation on the glutamatergic system. Immuhistochemical data revealed the presence of EAAT2 and EAAT3 surrounding noradrenergic neurons and EAAT2 on glial cells in the LC. CONCLUSIONS AND IMPLICATIONS: These results remark the importance of EAAT2 and EAAT3 in the regulation of rat LC by glutamate. Neuronal EAAT3 would be responsible for terminating the action of synaptically released glutamate, whereas glial EAAT2 would regulate tonic glutamate concentrations in this nucleus.

Caffeine-induced inhibition of the activity of glutamate transporter type 3 expressed in Xenopus oocytes.

Caffeine has been known to trigger seizures, however, the precise mechanism about the proconvulsive effect of caffeine remains unclear. Glutamate transporters play an important role to maintain the homeostasis of glutamate concentration in the brain tissue. Especially, dysfunction of excitatory amino acid transporter type 3 (EAAT3) can lead to seizures. We investigated the effects of caffeine on the activity of EAAT3 and the involvement of protein kinase C (PKC) and phosphatidylinositol 3-kinase (PI3K). Rat EAAT3 was expressed in Xenopus oocytes by injecting EAAT3 mRNA. l-Glutamate (30µM)-induced inward currents were recorded via the two-electrode voltage clamp method. Caffeine decreased EAAT3 activity in a dose-dependent manner. Caffeine (30µM for 3min) significantly reduced V(max), but did not alter K(m) value of EAAT3 for glutamate. When preincubated oocytes with phorbol-12-myristate-13-acetate (PMA, a PKC activator) were exposed to caffeine, PMA-induced increase in EAAT3 activity was abolished. Two PKC inhibitors (chelerythrine and staurosporine) significantly reduced basal EAAT3 activity. Whereas, there were no significant differences among the PKC inhibitors, caffeine, and PKC inhibitors+caffeine groups. In similarly fashion, wortmannin (a PI3K inhibitor) significantly decreased EAAT3 activity, however no statistical differences were observed among the wortmannin, caffeine, and wortmannin+caffeine groups. Our results demonstrate that caffeine attenuates EAAT3 activity and this reducing effect of caffeine seems to be mediated by PKC and PI3K.

Mechanism of inhibition of the glutamate transporter EAAC1 by the conformationally constrained glutamate analogue (+)-HIP-B.

Glutamate transporters play an important role in the regulation of extracellular glutamate concentrations in the mammalian brain and are, thus, promising targets for therapeutics. Despite this importance, the development of pharmacological tools has mainly focused on the synthesis of competitive inhibitors, which are amino acid analogues that bind to the substrate binding site. In this report, we describe the characterization of the mechanism of glutamate transporter inhibition by a constrained, cyclic glutamate analogue, (+)-3-hydroxy-4,5,6,6a-tetrahydro-3aH-pyrrolo[3,4-d]isoxazole-6-carboxylic acid [(+)-(3aS,6S,6aS)-HIP-B]. Our results show that (+)-HIP-B is a nontransportable amino acid that inhibits glutamate transporter function in a mixed mechanism. Although (+)-HIP-B inhibits the glutamate-associated anion conductance, it has no effect on the leak anion conductance, in contrast to competitive inhibitors. Furthermore, (+)-HIP-B is unable to alleviate the effect of the competitive inhibitor dl-threo-ß-benzyloxyaspartic acid (TBOA), which binds to the substrate binding site. (+)-HIP-B is more potent in inhibiting forward transport compared to reverse transport. In a mutant transporter, which is activated by glutamine, but not glutamate, (+)-HIP-B still acts as an inhibitor, although this mutant transporter is insensitive to TBOA. Finally, we analyzed the effect of (+)-HIP-B on the pre-steady-state kinetics of the glutamate transporter. The results can be explained with a mixed mechanism at a site that may be distinct from the substrate binding site, with a preference for the inward-facing configuration of the transporter and slow inhibitor binding. (+)-HIP-B may represent a new paradigm of glutamate transporter inhibition that is based on targeting of a regulatory site.

17ß-Estradiol attenuates the activity of the glutamate transporter type 3 expressed in Xenopus oocytes.

Estrogen, a neuroactive sex hormone in the brain, enhances neuronal excitability and increases seizures. Glutamate transporters help in limiting the excitatory neurotransmission by uptaking glutamate from the synapses. We investigated the effects of 17ß-estradiol on the activity of a glutamate transporter, excitatory amino acid transporter 3 (EAAT3), in Xenopus oocytes. EAAT3 was expressed in Xenopus oocytes by injection of rat EAAT3 mRNA. l-Glutamate (30 µM)-induced membrane currents mediated by EAAT3 were measured using the two-electrode voltage clamp technique. 17ß-Estradiol reduced EAAT3 activity in a concentration- and time-dependent manner. 17ß-Estradiol (10nM for 72h) significantly decreased V(max) but had no effect on K(m) of EAAT3 for glutamate. When 17ß-estradiol treated oocytes were incubated with phorbol-12-myrisate-13-acetate, a protein kinase C (PKC) activator, 17ß-estradiol-induced decrease in EAAT3 activity was abolished. Furthermore, in pretreatment of oocytes with chelerythrine or staurosporine, two PKC inhibitors, EAAT3 activity was significantly decreased. However, there was no statistical difference among the 17ß-estradiol, PKC inhibitor, or 17ß-estradiol plus PKC inhibitor groups. Likewise, wortmannin, a phosphatidylinositol 3-kinase (PI3K) inhibitor, significantly reduced basal EAAT3 activity, but the activity did not differ among the 17ß-estradiol, wortmannin, or 17ß-estradiol plus wortmannin groups. Estradiol receptor inhibitor, fulvestrant, did not change the reduced EAAT3 activity by 17ß-estradiol. Our results suggest that 17ß-estradiol decreases EAAT3 activity. PKC and PI3K seem to be involved in this effect, possibly not via estradiol receptors.

Inhibition of isoflurane-induced increase of cell-surface redistribution and activity of glutamate transporter type 3 by serine 465 sequence-specific peptides.

Excitatory amino acid transporters (EAAT) transport glutamate into cells to regulate glutamate neurotransmission and to maintain nontoxic extracellular glutamate levels for neurons. We showed previously that the commonly used volatile anesthetic isoflurane increases the transporting activity of EAAT3, the major neuronal EAAT. This effect requires a protein kinase C (PKC) α-mediated and S465-dependent EAAT3 redistribution to the plasma membrane. Thus, we hypothesize that specific peptides can be designed to block this effect. We conjugated a 10-amino acid synthetic peptide with a sequence identical to that of EAAT3 around the S465 to a peptide that can facilitate permeation of the plasma membrane. This fusion peptide inhibited the isoflurane-increased EAAT3 activity and redistribution to the plasma membrane in C6 cells and hippocampus. It did not affect the basal EAAT3 activity. This peptide also attenuated isoflurane-induced increase of PKCα in the immunoprecipitates produced by an anti-EAAT3 antibody. A scrambled peptide that has the same amino acid composition as the S465 sequence-specific peptide but has a random sequence did not change the effects of isoflurane on EAAT3. The S465 sequence-specific peptide, but not the scrambled peptide, is a good PKCα substrate in in vitro assay. These peptides did not affect cell viability. These results, along with our previous findings, strongly suggest that PKCα interacts with EAAT3 to regulate its functions. The S465 sequence-specific peptide may interrupt this interaction and is an effective inhibitor for the regulation of EAAT3 activity and trafficking by PKCα and isoflurane.