Alternate splicing and expression of the glutamate transporter EAAT5 in the rat retina

Excitatory amino acid transporter 5 (EAAT5) is an unusual glutamate transporter that is expressed in the retina, where it is localised to two populations of glutamatergic neurons, namely the bipolar neurons and photoreceptors. EAAT5 exhibits two distinct properties, acting both as a slow glutamate transporter and as a glutamate-gated inhibitory receptor. The latter property is attributable to a co-associated chloride conductance. EAAT5 has previously been thought to exist only as a full-length form. We now demonstrate by PCR cloning and sequencing, the presence of five novel splice variant forms of EAAT5 which skip either partial or complete exons in the rat retina. Furthermore, we demonstrate that each of these variants is expressed at the protein level as assessed by Western blotting using splice-specific antibodies that we have generated. We conclude that EAAT5 exists in multiple spliced forms, and propose, based upon retention or absence of key structural features, that these variant forms may potentially exhibit distinct properties relative to the originally described form of EAAT5.     

Abnormal exocytotic release of glutamate in a mouse model of amyotrophic lateral sclerosis

Glutamate-mediated excitotoxicity plays a major role in the degeneration of motor neurons in amyotrophic lateral sclerosis and reduced astrocytary glutamate transport, which in turn increases the synaptic availability of the amino acid neurotransmitter, was suggested as a cause. Alternatively, here we report our studies on the exocytotic release of glutamate as a possible source of excessive glutamate transmission. The basal glutamate efflux from spinal cord nerve terminals of mice-expressing human soluble superoxide dismutase (SOD1) with the G93A mutation [SOD1/G93A(+)], a transgenic model of amyotrophic lateral sclerosis, was elevated when compared with transgenic mice expressing the wild-type human SOD1 or to non-transgenic controls. Exposure to 15 mM KCl or 0.3 μM ionomycin provoked Ca(2+)-dependent glutamate release that was dramatically increased in late symptomatic and in pre-symptomatic SOD1/G93A(+) mice. Increased Ca(2+) levels were detected in SOD1/G93A(+) mouse spinal cord nerve terminals, accompanied by increased activation of Ca(2+)/calmodulin-dependent kinase II and increased phosphorylation of synapsin I. In line with these findings, release experiments suggested that the glutamate release augmentation involves the readily releasable pool of vesicles and a greater capability of these vesicles to fuse upon stimulation in SOD1/G93A(+) mice.     

岗田酸诱导大鼠脑神经细胞表达谷氨酸转运体EAAT1

为研究tau蛋白高度磷酸化与谷氨酸转运体功能之间的关系,实验采用免疫组织化学、荧光双标记技术及大鼠额叶皮质定位注射的方法,观察了蛋白磷酸酶抑制剂岗田酸（okadaic acid,OA)所致神经细胞退化对谷氨酸转运体亚型EAAT1表达的影响。结果如下：（1）在OA注射中心区神经元早期出现胞体固缩、肿胀、核移位,在注射3d时细胞破碎,发生坏死,并有大量炎性细胞浸润等病理现象;边周区细胞呈AT8（微管相关蛋白tau磷酸化指标）免疫阳性反应;（2）OA首先诱导神经细胞突起远端tau蛋白磷酸化,并逐渐向胞体发展,形成营养不良的神经细胞突起和神经纤维缠结样病理改变;（3）AT8免疫阳性反应脑区的神经细胞高表达谷氨酸转运体EAAT1,在12h阳性表达细胞数显著增多（P＜0．01）,1d时达峰值（P＜0．001）,3d时明显减少。在OA作用下EAAT1表达于星形胶质细胞和神经元。结果提示,OA致微管相关蛋白tau高度磷酸化时可诱导该区星形胶质细胞和神经元高表达谷氨酸转体EAAT1。EAAT1高表达的病理生理意义有待进一步的阐明。     

Expression of SOD1 G93A or wild-type SOD1 in primary cultures of astrocytes down-regulates the glutamate transporter GLT-1: lack of involvement of oxidative stress

Glutamate excitotoxicity is implicated in the aetiology of amyotrophic lateral sclerosis (ALS) with impairment of glutamate transport into astrocytes a possible cause of glutamate-induced injury to motor neurons. It is possible that mutations of Cu/Zn superoxide dismutase (SOD1), responsible for about 20% of familial ALS, down-regulates glutamate transporters via oxidative stress. We transfected primary mouse astrocytes to investigate the effect of the FALS-linked mutant hSOD1(G93A) and wild-type SOD1 (hSOD1wt) on the glutamate uptake system. Using western blotting, immunocytochemistry and RT-PCR it was shown that expression of either hSOD1(G93A) or hSOD1wt in astrocytes produced down-regulation of the levels of a glutamate transporter GLT-1, without alterations in its mRNA level. hSOD1(G93A) or hSOD1wt expression caused a decrease of the monomeric form of GLT-1 without increasing oxidative multimers of GLT-1. The effects were selective to GLT-1, since another glutamate transporter GLAST protein and mRNA levels were not altered. Reflecting the decrease in GLT-1 protein, [3H]d-aspartate uptake was reduced in cultures expressing hSOD1(G93A) or hSOD1wt. The hSOD1-induced decline in GLT-1 protein and [3H]d-aspartate uptake was not blocked by the antioxidant Trolox nor potentiated by antioxidant depletion using catalase and glutathione peroxidase inhibitors. Measurement of 2',7'-dichlorofluorescein (DCF)-induced fluorescence revealed that expression of hSOD1(G93A) or hSOD1wt in astrocytes does not lead to detectable increase of intracellular reactive oxygen species. This study suggests that levels of GLT-1 protein in astrocytes are reduced rapidly by overexpression of hSOD1, and is due to a property shared between the wild-type and G93A mutant form, but does not involve the production of intracellular oxidative stress.     

Critical amino acids in the TM2 of EAAT2 are essential for membrane-bound localization,substrate binding,transporter function and anion currents

Excitatory amino acid transporter 2 (EAAT2), the gene of which is known as solute carrier family 1 member 2 (SLC1A2), is an important membrane-bound transporter that mediates approximately 90% of the transport and clearance of l -glutamate at synapses in the central nervous system (CNS). Transmembrane domain 2 (TM2) of EAAT2 is close to hairpin loop 2 (HP2) and far away from HP1 in the inward-facing conformation. In the present study, 14 crucial amino acid residues of TM2 were identified via alanine-scanning mutations. Further analysis in EAAT2-transfected HeLa cells in vitro showed that alanine substitutions of these residues resulted in a decrease in the efficiency of trafficking/targeting to the plasma membrane and/or reduced functionality of membrane-bound, which resulted in impaired transporter activity. After additional mutations, the transporter activities of some alanine-substitution mutants recovered. Specifically, the P95A mutant decreased EAAT2-associated anion currents. The Michaelis constant (K_m) values of the mutant proteins L85A, L92A and L101A were increased significantly, whereas R87 and P95A were decreased significantly, indicating that the mutations L85A, L92A and L101A reduced the affinity of the transporter and the substrate, whereas R87A and P95A enhanced this affinity. The maximum velocity (Vmax) values of all 14 alanine mutant proteins were decreased significantly, indicating that all these mutations reduced the substrate transport rate. These results suggest that critical residues in TM2 affect not only the protein expression and membrane-bound localization of EAAT2, but also its interactions with substrates. Additionally, our findings elucidate that the P95A mutant decreased EAAT2-related anion currents. Our results indicate that the TM2 of EAAT2 plays a vital role in the transport process. The key residues in TM2 affect protein expression in the membrane, substrate transport and the anion currents of EAAT2.     

Loss of metabotropic glutamate receptor-mediated regulation of glutamate transport in chemically activated astrocytes in a rat model of amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by a selective loss of motor neurones accompanied by intense gliosis in lesioned areas of the brain and spinal cord. Glutamate-mediated excitotoxicity resulting from impaired astroglial uptake constitutes one of the current pathophysiological hypotheses explaining the progression of the disease. In this study, we examined the regulation of glutamate transporters by type 5 metabotropic glutamate receptor (mGluR5) in activated astrocytes derived from transgenic rats carrying an ALS-related mutated human superoxide dismutase 1 (hSOD1(G93A)) transgene. Cells from transgenic animals and wild-type littermates showed similar expression of glutamate-aspartate transporter and glutamate transporter 1 (GLT-1) after in vitro activation, whereas cells carrying the hSOD1 mutation showed a three-fold higher expression of functional mGluR5, as observed in the spinal cord of end-stage animals. In cells from wild-type animals, (S)-3,5-dihydroxyphenylglycine (DHPG) caused an immediate protein kinase C (PKC)-dependent up-regulation of aspartate uptake that reflected the activation of GLT-1. Although this effect was mimicked in both cultures by direct activation of PKC using phorbol myristate acetate, DHPG failed to up-regulate aspartate uptake in cells derived from the transgenic rats. The failure of activated mGluR5 to increase glutamate uptake in astrocytes derived from this animal model of ALS supports the theory of glutamate excitotoxicity in the pathogenesis of the disease.     

Structure-activity relationship study of pyridazine derivatives as glutamate transporter EAAT2 activators

Excitatory amino acid transporter 2 (EAAT2) is the major glutamate transporter and functions to remove glutamate from synapses. A thiopyridazine derivative has been found to increase EAAT2 protein levels in astrocytes. A structure-activity relationship study revealed that several components of the molecule were required for activity, such as the thioether and pyridazine. Modification of the benzylthioether resulted in several derivatives (7-13, 7-15 and 7-17) that enhanced EAAT2 levels by >6-fold at concentrations < 5 μM after 24h. In addition, one of the derivatives (7-22) enhanced EAAT2 levels 3.5-3.9-fold after 24h with an EC(50) of 0.5 μM.     

Alternative non-coding exons support serotonin transporter mRNA expression in the brain and gut

A number of studies in recent years have linked polymorphisms within the serotonin transporter (5HTT) gene to affective disorders and anxiety traits. The human 5HTT mRNA is alternatively spliced, and the splice variants are equally expressed in the human placental cell line and dorsal raphe. In this study, using 5' rapid amplification of cDNA ends, we show that the rat 5HTT mRNA is alternatively spliced, leading to three distinct mRNAs differing in the 5' untranslated region. To determine whether the three alternatively spliced mRNA species that contain one of the following untranslated regions (i) exon 1A, 63 bp (ii) exon 1A + 1B, 125 bp or (iii) exon 1C, 101 bp, were expressed in a tissue-specific manner, we used RT-PCR and exon-specific oligonucleotide hybridization. Our results suggest two of the variants (1A + 1B and 1A) may utilize the same promoter; however, they are not equally expressed. While in the adult CNS and adrenal medulla, the shorter mRNA consisting of exon 1A was considerably more abundant, in the stomach and heart, the two variants were equally expressed. The third splice variant exon 1C is only expressed in the gut and to a lesser extent in the heart. The data from this study suggest the splice variant consisting of exon 1C may utilize a distinct promoter compared to the other two.     

Behavioural effects of cage systems on the G93A Superoxide Dismutase 1 transgenic mouse model for amyotrophic lateral sclerosis

Environmental factors inherent to animal facilities can impact on the neuro-behavioural phenotype of laboratory mice and genetic mouse models for human diseases. Many facilities have upgraded from traditional ‘open filter top’ cages (FT) to individually ventilated cage (IVC) systems, which have been shown to modify various behavioural responses of laboratory mice. Importantly, the impact of IVC housing on the G93A superoxide dismutase 1 mouse model of amyotrophic lateral sclerosis (ALS) is currently unknown. Male and female wild type-like (WT) and heterozygous SOD1^G93A mice were group-housed in FT or IVC systems from PND 30 ± 5 onwards. Body weight and motor function were assessed weekly from 15 weeks onward. Mice were also tested for cognitive abilities (i.e., fear conditioning and social recognition memory) and sensorimotor gating (i.e., prepulse inhibition: PPI). SOD1^G93A mice lost body weight, and their motor function degenerated over time compared with control littermates. Motor impairments developed faster when SOD1^G93A females were housed in IVCs. Context and cue freezing were increased in SOD1^G93A females compared with controls, whereas all SOD1^G93A mice exhibited lower acoustic startle and PPI than WT mice. IVC housing led to an increase in cue freezing in males and reduced the severity of PPI deficits in SOD1^G93A females. Overall, IVC housing impacted moderately on the SOD1^G93A phenotype but central behavioural deficits were still evident across housing conditions. Nonetheless, our findings indicate the importance of assessing the effect of cage system in genetic mouse models as these systems can modulate the magnitude and onset of genotypic differences.     

Inducible superoxide dismutase 1 aggregation in transgenic amyotrophic lateral sclerosis mouse fibroblasts

High molecular weight detergent-insoluble complexes of superoxide dismutase 1 (SOD1) enzyme are a biochemical abnormality associated with mutant SOD1-linked familial amyotrophic lateral sclerosis (FALS). In the present study, SOD1 protein from spinal cords of transgenic FALS mice was fractionated according to solubility in saline, zwitterionic, non-ionic or anionic detergents. Both endogenous mouse SOD1 and mutant human SOD1 were least soluble in SDS, followed by NP-40 and CHAPS, with an eight-fold greater detergent resistance of mutant protein overall. Importantly, high molecular weight mutant SOD1 complexes were isolated with SDS-extraction only. To reproduce SOD1 aggregate pathology in vitro, primary fibroblasts were isolated and cultured from neonatal transgenic FALS mice. Fibroblasts expressed abundant mutant SOD1 without spontaneous aggregation over time with passage. Proteasomal inhibition of cultures using lactacystin induced dose-dependent aggregation and increased the SDS-insoluble fraction of mutant SOD1, but not endogenous SOD1. In contrast, paraquat-mediated superoxide stress in fibroblasts promoted aggregation of endogenous SOD1, but not mutant SOD1. Treatment of cultures with peroxynitrite or the copper chelator diethyldithiocarbamate (DDC) alone did not modulate aggregation. However, DDC inhibited lactacystin-induced mutant SOD1 aggregation in transgenic fibroblasts, while exogenous copper slightly augmented aggregation. These data suggest that SOD1 aggregates may derive from proteasomal or oxidation-mediated oligomerisation pathways from mutant and endogenous subunits respectively. Furthermore, these pathways may be affected by copper availability. We propose that non-neural cultures such as these transgenic fibroblasts with inducible SOD1 aggregation may be useful for rapid screening of compounds with anti-aggregation potential in FALS.     

Cooperative action of glutamate transporters and cystine/glutamate antiporter system Xc- protects from oxidative glutamate toxicity

Oxidative glutamate toxicity in the neuronal cell line HT22 is a model for cell death by oxidative stress. In this paradigm, an excess of extracellular glutamate blocks the glutamate/cystine-antiporter system Xc-, depleting the cell of cysteine, a building block of the antioxidant glutathione. Loss of glutathione leads to the accumulation of reactive oxygen species and eventually cell death. We selected cells resistant to oxidative stress, which exhibit reduced glutamate-induced glutathione depletion mediated by an increase in the antiporter subunit xCT and system Xc- activity. Cystine uptake was less sensitive to inhibition by glutamate and we hypothesized that glutamate import via excitatory amino acid transporters and immediate re-export via system Xc- underlies this phenomenon. Inhibition of glutamate transporters by l-trans-pyrrolidine-2,4-dicarboxylic acid (PDC) and DL-threo-beta-benzyloxyaspartic acid (TBOA) exacerbated glutamate-induced cell death. PDC decreased intracellular glutamate accumulation and exacerbated glutathione depletion in the presence of glutamate. Transient overexpression of xCT and the glutamate transporter EAAT3 cooperatively protected against glutamate. We conclude that EAATs support system Xc- to prevent glutathione depletion caused by high extracellular glutamate. This knowledge could be of use for the development of novel therapeutics aimed at diseases associated with depletion of glutathione like Parkinson's disease.     

EAAT4 phosphorylation at the SGK1 consensus site is required for transport modulation by the kinase

EAAT4 (SLC1A6) is a Purkinje-Cell-specific post-synaptic excitatory amino acid transporter that plays a major role in clearing synaptic glutamate. EAAT4 abundance and function is known to be modulated by the serum and glucocorticoid inducible kinase (SGK) 1 but the precise mechanism of kinase action has not been defined yet. The present work aims to identify the molecular mechanism of EAAT4 modulation by the kinase. The EAAT4 sequence bears two putative SGK1 consensus sites (at Thr40 and Thr504) at the amino and carboxy terminus that are conserved among species. Expression studies in Xenopus oocytes demonstrated that EAAT4-mediated [(3)H] glutamate uptake and cell surface abundance are enhanced by co-expression of SGK1. Disruption of the SGK1 phosphorylation site at threonine 40 ((T40A)EAAT4) or of both phosphorylation sites ((T40AT504A)EAAT4) abrogated the effect of SGK1 on transporter function and expression. SGK1 modulates several transport proteins via inhibition of the ubiquitin ligase Nedd4-2. Co-expression of Nedd4-2 inhibited wild-type EAAT4 but not the (T40AT504A)EAAT4 mutant. Besides, RNA interference-mediated reduction of endogenous Nedd4-2 (xNedd4-2) expression increased the activity of the transporter. In conclusion, maximal glutamate transport modulation by SGK1 is accomplished by direct EAAT4 stimulation and to a lesser extent by inhibition of intrinsic Nedd4-2.     

Molecular determinants of transport stimulation of EAAT2 are located at interface between the trimerization and substrate transport domains

Excitatory amino acid transporters (EAATs) regulate glutamatergic signal transmission by clearing extracellular glutamate. Dysfunction of these transporters has been implicated in the pathogenesis of various neurological disorders. Previous studies have shown that venom from the spider Parawixia bistriata and a purified compound (Parawixin1) stimulate EAAT2 activity and protect retinal tissue from ischemic damage. In the present study, the EAAT2 subtype specificity of this compound was explored, employing chimeric proteins between EAAT2 and EAAT3 transporter subtypes and mutants to characterize the structural region targeted by the compound. This identified a critical residue (Histidine‐71 in EAAT2 and Serine‐45 in EAAT3) in transmembrane domain 2 (TM2) to be important for the selectivity between EAAT2 and EAAT3 and for the activity of the venom. Using the identified residue in TM2 as a structural anchor, several neighboring amino acids within TM5 and TM8 were identified to also be important for the activity of the venom. This structural domain of the transporter lies at the interface of the rigid trimerization domain and the central substrate‐binding transport domain. Our studies suggest that the mechanism of glutamate transport enhancement involves an interaction with the transporter that facilitates the movement of the transport domain.

     

Differential expression of inflammation- and apoptosis-related genes in spinal cords of a mutant SOD1 transgenic mouse model of familial amyotrophic lateral sclerosis

Familial amyotrophic lateral sclerosis (FALS)-linked mutations in copper-zinc superoxide dismutase (SOD1) cause motor neuron death through one or more acquired toxic properties. We analyzed the molecular mechanism underlying motor neuron degeneration in the transgenic mouse model expressing the SOD1 gene with G93A mutation. Using cDNA microarray, the differentially expressed genes were identified in the spinal cords of G93A mice, 30 being elevated and seven decreased. cDNA microarray analysis to monitor gene expression during neurodegeneration revealed an up-regulation of genes related to an inflammatory process, such as the tumor necrosis factor-alpha (TNF-alpha) gene, resulting from glial cell activation, together with the change in apoptosis-related gene expression, such as caspase-1. The increased expression of the inflammation- and apoptosis-related genes occurred at 11 weeks of age in the presymptomatic stage prior to motor neuron death. These results suggest a mechanism of neurodegeneration that includes an inflammatory response as an important component. Thus, ALS has paralleled other neurodegenerative disorders, such as Alzheimer's and prion diseases, in which the inflammatory process is believed to participate directly in neuronal death.     

Cancer cells release glutamate via the cystine/glutamate antiporter

Although the amino acid glutamate is used as an intercellular signaling molecule for normal bone homeostasis, little is known regarding its possible role in the metabolic disruption characteristic of bone metastasis. We have previously shown in vitro that cancer cell lines relevant to bone metastasis release glutamate into the extracellular environment. This study demonstrates the expression of multiple glutamate transporters in cancer cell lines of non-central nervous system origin. Furthermore, we identify the molecular mechanism responsible for glutamate export and show that this system can be inhibited pharmacologically. By highlighting that glutamate secretion is a common biological feature of cancer cells, this study suggests that tumor-derived glutamate could interfere with glutamate-dependent intercellular signaling in normal bone. Pharmacological interference with cancer cell glutamate release may be a viable option for limiting host bone response to invading tumor cells in bone metastasis.     

Informatics-assisted protein profiling in a transgenic mouse model of amyotrophic lateral sclerosis

One of the causes of amyotrophic lateral sclerosis (ALS) is due to mutations in Cu,Zn-superoxide dismutase (SOD1). The mutant protein exhibits a toxic gain of function that adversely affects the function of neurons in the spinal cord, brain stem, and motor cortex. A proteomic analysis of protein expression in a widely used mouse model of ALS was undertaken to identify differences in protein expression in the spinal cords of mice expressing a mutant protein with the G93A mutation found in human ALS. Protein profiling was done on soluble and particulate fractions of spinal cord extracts using high throughput two-dimensional liquid chromatography coupled to tandem mass spectrometry. An integrated proteomics-informatics platform was used to identify relevant differences in protein expression based upon the abundance of peptides identified by database searching of mass spectrometry data. Changes in the expression of proteins associated with mitochondria were particularly prevalent in spinal cord proteins from both mutant G93A-SOD1 and wild-type SOD1 transgenic mice. G93A-SOD1 mouse spinal cord also exhibited differences in proteins associated with metabolism, protein kinase regulation, antioxidant activity, and lysosomes. Using gene ontology analysis, we found an overlap of changes in mRNA expression in presymptomatic mice (from microarray analysis) in three different gene categories. These included selected protein kinase signaling systems, ATP-driven ion transport, and neurotransmission. Therefore, alterations in selected cellular processes are detectable before symptomatic onset in ALS mouse models. However, in late stage disease, mRNA expression analysis did not reveal significant changes in mitochondrial gene expression but did reveal concordant changes in lipid metabolism, lysosomes, and the regulation of neurotransmission. Thus, concordance of proteomic and mRNA expression data within multiple categories validates the use of gene ontology analysis to compare different types of "omic" data.     

Bax and Bcl-2 interaction in a transgenic mouse model of familial amyotrophic lateral sclerosis

It has been proposed that mutations in copper/zinc-superoxide dismutase (SOD1), the only proven cause of amyotrophic lateral sclerosis (ALS), induce the disease by a toxic property that promotes apoptosis. Consistent with this, we have demonstrated that overexpression of Bcl-2, a protein that inhibits apoptosis, attenuates neurodegeneration produced by the familial ALS-linked SOD1 mutant G93A (mSOD1). Herein, we assessed the status of key members of the Bcl-2 family in the spinal cord of transgenic mSOD1 mice at different stages of the disease. In asymptomatic transgenic mSOD1 mice, expression of Bcl-2, Bcl-XL, Bad, and Bax does not differ from that in nontransgenic mice. In contrast, in symptomatic mice, expression of Bcl-2 and Bcl-XL, which inhibit apoptosis, is reduced, whereas expression of Bad and Bax, which stimulate apoptosis, is increased. These alterations are specific to affected brain regions and are caused by the mutant and not by the normal SOD1 enzyme. Relevant to the neuroprotective effects of Bcl-2 in transgenic mSOD1 mice, overexpression of Bcl-2 increases the formation of Bcl-2:Bax heterodimers, which abolish the Bax proapoptotic property. This study demonstrates significant alterations in the expression of key members of the Bcl-2 family associated with mSOD1 deleterious effects. That these changes contribute to the neurodegenerative process in this model of ALS is supported by our observations in double transgenic mSOD1/Bcl-2 mice in which the pernicious increase of Bax is tempered by an increase in formation of Bcl-2:Bax heterodimers. Based on these findings, it may be concluded that Bcl-2 family members appear as invaluable targets for the development of new neuroprotective therapies in ALS.     

Effects of threo-β-hydroxyaspartate derivatives on excitatory amino acid transporters (EAAT4 and EAAT5)

D,L-threo-beta-Benzyloxyaspartate (D,L-TBOA), an analog of threo-beta-hydroxyaspartate (THA) possessing a bulky substituent, is a potent non-transportable blocker for the excitatory amino acid transporters, EAAT1, 2 and 3, while L-threo-beta-methoxyaspartate (L-TMOA) is a blocker for EAAT2, but a substrate for EAAT1 and EAAT3. To characterize the actions of these THA analogs and the function of EAAT4 and EAAT5, we performed electrophysiological analyses in EAAT4 or EAAT5 expressed on Xenopus oocytes. In EAAT4-expressing oocytes, D,L-TBOA acted as a non-transportable blocker, while L-TMOA like D,L-THA was a competitive substrate. In contrast, D,L-THA, D,L-TBOA and L-TMOA all strongly attenuated the glutamate-induced currents generated by EAAT5. Among them, L-TMOA showed the most potent inhibitory action. Moreover, D,L-THA, D,L-TBOA and L-TMOA themselves elicited outward currents at negative potentials and remained inward at positive potentials suggesting that D,L-TBOA and L-TMOA, as well as D,L-THA, not only act as non-transportable blockers, but also block the EAAT5 leak currents. These results indicate that EAATs 4 and 5 show different sensitivities to THA analogs although they share properties of a glutamate-gated chloride channel.