Excitatory Amino Acid Transporter EAAT3 Research Articles

Altered Grooming Syntax and Amphetamine-Induced Dopamine Release in EAAT3 Overexpressing Mice.

The excitatory amino acid transporter EAAT3 plays an important role in the neuronal uptake of glutamate regulating the activation of glutamate receptors. Polymorphisms in the gene-encoding EAAT3 have been associated with obsessive–compulsive disorder (OCD), although the mechanisms underlying this relationship are still unknown. We recently reported that mice with increased EAAT3 expression in forebrain neurons (EAAT3glo/CMKII) display behavioral and synaptic features relevant to OCD, including increased grooming, higher anxiety-like behavior and altered cortico-striatal synaptic function. The dopamine neurotransmitter system is implicated in ritualistic behaviors. Indeed, dopaminergic neurons express EAAT3, and mice lacking EAAT3 exhibit decreased dopamine release and decreased expression of the dopamine D1 receptor. Moreover, EAAT3 plays a role on the effect of the psychostimulant amphetamine. As such, we sought to determine if the OCD-like behavior in EAAT3glo/CMKII mice is accompanied by altered nigro-striatal dopaminergic transmission. The aim of this study was to analyze dopamine transmission both in basal conditions and after an acute challenge of amphetamine, using behavioral, neurochemical, molecular, and cellular approaches. We found that in basal conditions, EAAT3glo/CMKII mice performed more grooming events and that they remained in phase 1 of the grooming chain syntax compared with control littermates. Administration of amphetamine increased the number of grooming events in control mice, while EAAT3glo/CMKII mice remain unaffected. Interestingly, the grooming syntax of amphetamine-control mice resembled that of EAAT3glo/CMKII mice in basal conditions. Using in vivo microdialysis, we found decreased basal dopamine levels in EAAT3glo/CMKII compared with control mice. Unexpectedly, we found that after acute amphetamine, EAAT3glo/CMKII mice had a higher release of dopamine compared with that of control mice, suggesting that EAAT3 overexpression leads to increased dopamine releasability. To determine postsynaptic effect of EAAT3 overexpression over dopamine transmission, we performed Western blot analysis of dopaminergic proteins and found that EAAT3glo/CMKII mice have higher expression of D2 receptors, suggesting a higher inhibition of the indirect striatal pathway. Together, the data indicate that EAAT3 overexpression impacts on dopamine transmission, making dopamine neurons more sensitive to the effect of amphetamine and leading to a disbalance between the direct and indirect striatal pathways that favors the performance of repetitive behaviors.

Neuronal excitatory amino acid transporter EAAT3: Emerging functions in health and disease

Plasma membrane neurotransmitter transporters maintain extracellular concentrations of neurotransmitters by facilitating transport into the cytosol. This regulation of extracellular neurotransmitters limits binding to receptors and activation of downstream signaling pathways. In addition to this critical function, transporters modulate neuronal activity via direct gating of transporter-associated ion channels and indirectly through trafficking of transporters to and from the plasma membrane. These functions are dependent on diverse expression patterns and levels of the transporters throughout the brain. This diversity is highlighted within the excitatory amino acid transporter family, which consists of five excitatory amino acid transporters found in the mammalian central nervous system (CNS). EAAT1 (GLAST) and EAAT2 (GLT-1) are primarily expressed in astrocytes while EAAT3 expression is mainly observed in many neurons throughout the brain (Holmseth et al., 2012). In contrast, EAAT4 is most prominently expressed in cerebellar Purkinje neurons and EAAT5 is exclusively found in the retina. In general, the astroglial transporters are highly expressed in the brain; EAAT2 is the most abundant, followed by EAAT1 with approximately a 4-fold lower expression (Holmseth et al., 2012). High expression levels of these glial transporters are consistent with their role in glutamate clearance (Lehre and Danbolt, 1998; Rothstein et al., 1996; Tanaka et al., 1997). The role of the neuronal transporter EAAT3 in brain has been more difficult to elucidate. Levels of EAAT3 are approximately 100-fold lower than EAAT2 (Holmseth et al., 2012) but EAAT3 expression is observed throughout the CNS, with enriched expression in the cerebral cortex, hippocampus, cerebellum and basal ganglia (Rothstein et al., 1994; Shashidharan et al., 1997). Given the lack of selective EAAT3 inhibitors, studies have relied on EAAT3 transporters expressed in various cells and endogenous transporters expressed in cultured hippocampal neurons (Diamond and Jahr, 1997; Grewer et al., 2000; Wadiche et al., 1995b), as well as EAAT3 knockout mice (Scimemi et al., 2009) to dertmine the physiological functions of EAAT3. These studies determined that the time course of glutamate in the synaptic cleft is a function of the binding of glutamate to EAATs and that transport of glutamate does not significantly contribute to the amplitude or kinetics of synaptic responses due to the relatively slow transport cycle (Diamond and Jahr, 1997; Tong and Jahr, 1994; Wadiche and von Gersdorff, 2006). Interestingly, EAAT3 knockout mice exhibit few behavioral deficits (Peghini et al., 1997), and antisense oligonucleotide knockdown in the striatum results in minimal elevation of extracellular glutamate levels or neurodegeneration, in contrast to knockdown of EAATs 1 and 2 (Rothstein et al., 1996). The lack of neurodegeneration is particularly surprising given that EAAT3 also serves as a cysteine transporter (Aoyama et al., 2006; Watts et al., 2014; Zerangue and Kavanaugh, 1996). Cysteine is the rate-limiting substrate for the synthesis of the antioxidant glutathione and its extracellular depletion is hypothesized to contribute to neurodegeneration. EAAT3 is also the dominant glutamate transporter in the intestines and provides nutrient absorption from the diet (Hu et al., 2018), but knockout animals appear to grow at a comparable rate to their litter mates (Peghini et al., 1997). The most remarkable initial observation from EAAT3 knockout mice was aminoaciduria due to the absence of EAAT3 in the kidneys (Peghini et al., 1997). The reported lack of overt behavioral abnormalities in the EAAT3 knockout mice suggest that either EAAT3 is not integral to regulation of glutamatergic signaling in the brain or that substantial developmental compensatory changes are induced in these mice. Human genetic studies and constitutive deletion mouse models have now provided evidence that EAAT3 has important roles in regulating neuronal signaling.

Up-Regulation of Excitatory Amino Acid Transporters EAAT3 and EAAT4 by Lithium Sensitive Glycogen Synthase Kinase GSK3ß

Background: Cellular uptake of glutamate by the excitatory amino-acid transporters (EAATs) decreases excitation and thus participates in the regulation of neuroexcitability. Kinases impacting on neuronal function include Lithium-sensitive glycogen synthase kinase GSK3ß. The present study thus explored whether the activities of EAAT3 and/or EAAT4 isoforms are sensitive to GSK3ß. Methods: cRNA encoding wild type EAAT3 (SLC1A1) or EAAT4 (SLC1A6) was injected into Xenopus oocytes without or with additional injection of cRNA encoding wild type GSK3ß or the inactive mutant ^K85AGSK3ß. Dual electrode voltage clamp was performed in order to determine glutamate-induced current (I_EAAT). Results: Appreciable I_EAAT was observed in EAAT3 or EAAT4 expressing but not in water injected oocytes. I_EAAT was significantly increased by coexpression of GSK3ß but not by coexpression of ^K85AGSK3ß. Coexpression of GSK3ß increased significantly the maximal I_EAAT in EAAT3 or EAAT4 expressing oocytes, without significantly modifying apparent affinity of the carriers. Lithium (1 mM) exposure for 24 hours decreased I_EAAT in EAAT3 and GSK3ß expressing oocytes to values similar to I_EAAT in oocytes expressing EAAT3 alone. Lithium did not significantly modify I_EAAT in oocytes expressing EAAT3 without GSK3ß. Conclusions: Lithium-sensitive GSK3ß is a powerful regulator of excitatory amino acid transporters EAAT3 and EAAT4.

SPAK and OSR1 Sensitivity of Excitatory Amino Acid Transporter EAAT3

Background/Aims: Kinases involved in the regulation of epithelial transport include SPAK (SPS1-related proline/alanine-rich kinase) and OSR1 (oxidative stress-responsive kinase 1). SPAK and OSR1 are both regulated by WNK (with-no-K(Lys)) kinases. The present study explored whether SPAK and/or OSR1 influence the excitatory amino acid transporter EAAT3, which accomplishes glutamate and aspartate transport in kidney, intestine and brain. Methods: cRNA encoding EAAT3 was injected into Xenopus laevis oocytes with or without additional injection of cRNA encoding wild-type SPAK, constitutively active ^T233ESPAK, WNK insensitive ^T233ASPAK, catalytically inactive ^D212ASPAK, wild-type OSR1, constitutively active ^T185EOSR1, WNK insensitive ^T185AOSR1 and catalytically inactive ^D164AOSR1. Glutamate-induced current was taken as measure of electrogenic glutamate transport and was quantified utilizing dual electrode voltage clamp. Furthermore, Ussing chamber was employed to determine glutamate transport in the intestine from gene-targeted mice carrying WNK insensitive SPAK (spak^tg/tg) and from corresponding wild-type mice (spak^+/+). Results: EAAT3 activity was significantly decreased by wild-type SPAK and ^T233ESPAK, but not by ^T233ASPAK and ^D212ASPAK. SPAK decreased maximal transport rate without affecting significantly affinity of the carrier. Similarly, EAAT3 activity was significantly downregulated by wild-type OSR1 and ^T185EOSR1, but not by ^T185AOSR1 and ^D164AOSR1. Again OSR1 decreased maximal transport rate without affecting significantly affinity of the carrier. Intestinal electrogenic glutamate transport was significantly lower in spak^+/+ than in spak^tg/tg mice. Conclusion: Both, SPAK and OSR1 are negative regulators of EAAT3 activity.

Regulation of cellular transport by klotho protein.

The antiaging protein of Klotho is a transmembrane protein mainly expressed in the kidney, parathyroid glands and choroid plexus of the brain. The Klotho protein exists in two forms, a full-length membrane form and a soluble secreted form. The extracellular domain of Klotho can be enzymatically cleaved off and released into the systemic circulation where it acts as β-glucuronidase and a hormone. Soluble Klotho can be found in the blood, cerebrospinal fluid, and the urine of mammals. Klotho deficiency results in early appearance of multiple age-related disorders and premature death, whereas overexpression of Klotho exerts the opposite effect. Klotho may influence cellular transport processes across the cell membrane by inhibiting calcitriol (1,25(OH) (2)D(3)), formation or by directly affecting transporter proteins, including ion channels, carriers and pumps. Accordingly, Klotho protein is a powerful regulator of transport mechanisms across the cell membrane. Klotho regulates diverse calcium and potassium ion channels, as well as several carriers including the Na(+)-coupled excitatory amino acid transporters EAAT3 and EAAT4, the Na(+)-coupled phosphate cotransporters, NaPi-IIa and NaPi-IIb, and a Na(+)/K(+)-ATPase. All those cellular transport regulations contribute in the aging suppressor role of Klotho. Future studies will help to determine if the Klotho protein regulates cell-surface expression of other transport proteins and is affecting underlying mechanisms.

Klotho Sensitivity of the Neuronal Excitatory Amino Acid Transporters EAAT3 and EAAT4

Klotho, a transmembrane protein, which can be cleaved off as β-glucuronidase and hormone, is released in both, kidney and choroid plexus and encountered in blood and cerebrospinal fluid. Klotho deficiency leads to early appearance of age-related disorders and premature death. Klotho may modify transport by inhibiting 1,25(OH)2D3 formation or by directly affecting channel and carrier proteins. The present study explored whether Klotho influences the activity of the Na+-coupled excitatory amino acid transporters EAAT3 and EAAT4, which are expressed in kidney (EAAT3), intestine (EAAT3) and brain (EAAT3 and EAAT4). To this end, cRNA encoding EAAT3 or EAAT4 was injected into Xenopus oocytes with and without additional injection of cRNA encoding Klotho. EAAT expressing Xenopus oocytes were further treated with recombinant human β-Klotho protein with or without β-glucuronidase inhibitor D-saccharic acid 1,4-lactone monohydrate (DSAL). Electrogenic excitatory amino acid transport was determined as L-glutamate-induced current (Iglu) in two electrode voltage clamp experiments. EAAT3 and EAAT4 protein abundance in the Xenopus oocyte cell membrane was visualized by confocal microscopy and quantified utilizing chemiluminescence. As a result, coexpression of Klotho cRNA significantly increased Iglu in both, EAAT3 or EAAT4-expressing Xenopus oocytes. Klotho cRNA coexpression significantly increased the maximal current and cell membrane protein abundance of both EAAT3 and EAAT4. The effect of Klotho coexpression on EAAT3 and EAAT4 activity was mimicked by treating EAAT3 or EAAT4-expressing Xenopus oocytes with recombinant human β-Klotho protein. The effects of Klotho coexpression and of treatment with recombinant human β-Klotho protein were both abrogated in the presence of DSAL (10 µM). In conclusion, Klotho is a novel, powerful regulator of the excitatory amino acid transporters EAAT3 and EAAT4.

Opposite Movements of the External Gate in Glutamate Transporters upon Binding Different Cotransported Ligands Measured by EPR

Hairpin two (HP2) has been proposed as the extracellular gate of glutamate transporters. To test this hypothesis, we use double site-directed spin-labeling electron paramagnetic resonance spectroscopy on the bacterial transporter GltPh to examine conformational changes in HP2. Surprisingly, the two co-ligands Na+ and aspartate induce opposite movements of HP2. We find that Na+ binding to the apo state of the transporter opens the extracellular gate, while the subsequent binding of aspartate closes the gate. In addition, using voltage clamp fluorometry on the mammalian excitatory amino acid transporter EAAT3, we confirm that the opposite conformational changes of HP2 induced by Na+ and amino acid substrates also occur in mammalian amino acid transporters. Our findings are consistent with HP2 comprising the extracellular gate of glutamate transporters, and that Na+ binding opens and stabilizes the extracellular gate thereby allowing for amino acid substrate binding.

PIP5K2A-dependent regulation of excitatory amino acid transporter EAAT3

According to previous observations, the gene encoding the phosphatidylinositol-4-phosphate 5-kinase II alpha (PIP5K2A) is associated with schizophrenia. Specifically, the mutation (N251S)PIP5K2A has been discovered in schizophrenic patients but not in healthy individuals. A defect of the excitatory amino acid transporter EAAT3 has similarly been implicated in the development of schizophrenia. The present study thus explored whether PIP5K2A is involved in the regulation of EAAT3 activity. EAAT3 was expressed in Xenopus oocytes either without or with PIP5K2A, and EAAT3 transporter activity was estimated from the glutamate (2-mM)-induced current (I(glu)) in dual electrode voltage clamp experiments. EAAT3 protein abundance in the cell membrane was estimated by Western blotting and confocal microscopy. In EAAT3-expressing oocytes, I(glu) was enhanced by coexpression of wild type PIP5K2A. Coexpression of the schizophrenia-associated mutant (N251S)PIP5K2A significantly decreased I(glu) in oocytes expressing EAAT3 with or without additional expression of wild type PIP5K2A. Thus, (N251S)PIP5K2A exerts a dominant inhibitory effect. Membrane abundance of EAAT3 was increased by wild type PIP5K2A and decreased by (N251S)PIP5K2A in both EAAT3-expressing oocytes and human embryonic kidney cells. The present observations disclose a novel mechanism of EAAT3 regulation, which may contribute to the deranged regulation of excitability in schizophrenic patients.

The substituted aspartate analogue l-β- threo-benzyl-aspartate preferentially inhibits the neuronal excitatory amino acid transporter EAAT3

The excitatory amino acid transporters (EAATs) play key roles in the regulation of CNS l-glutamate, especially related to synthesis, signal termination, synaptic spillover, and excitotoxic protection. Inhibitors available to delineate EAAT pharmacology and function are essentially limited to those that non-selectively block all EAATs or those that exhibit a substantial preference for EAAT2. Thus, it is difficult to selectively study the other subtypes, particularly EAAT1 and EAAT3. Structure activity studies on a series of β-substituted aspartate analogues identify l-β-benzyl-aspartate ( l-β-BA) as among the first blockers that potently and preferentially inhibits the neuronal EAAT3 subtype. Kinetic analysis of d-[ 3H]aspartate uptake into C17.2 cells expressing the hEAATs demonstrate that l-β- threo-BA is the more potent diastereomer, acts competitively, and exhibits a 10-fold preference for EAAT3 compared to EAAT1 and EAAT2. Electrophysiological recordings of EAAT-mediated currents in Xenopus oocytes identify l-β-BA as a non-substrate inhibitor. Analyzing l-β- threo-BA within the context of a novel EAAT2 pharmacophore model suggests: (1) a highly conserved positioning of the electrostatic carboxyl and amino groups; (2) nearby regions that accommodate select structural modifications (cyclopropyl rings, methyl groups, oxygen atoms); and (3) a unique region l-β- threo-BA occupied by the benzyl moieties of l-TBOA, l-β- threo-BA and related analogues. It is plausible that the preference of l-β- threo-BA and l-TBOA for EAAT3 and EAAT2, respectively, could reside in the latter two pharmacophore regions.

Molecular cloning, gene structure, expression profile and functional characterization of the mouse glutamate transporter (EAAT3) interacting protein GTRAP3–18

Glutamate is an important amino acid implicated in energy metabolism, protein biosynthesis and neurotransmission. The Na+-dependent high-affinity excitatory amino acid transporter EAAT3 (EAAC1) facilitates glutamate uptake into most cells. Recently, a novel rat EAAT3-interacting protein called GTRAP3–18 has been identified by a yeast two-hybrid screening. GTRAP3–18 functions as a negative modulator of EAAT3-mediated glutamate transport. In order to further understand the function and regulation of GTRAP3–18, we cloned the mouse orthologue to GTRAP3–18 and determined its gene structure and its expression pattern. GTRAP3–18 encodes a 188-residue hydrophobic protein whose sequence is highly conserved amongst vertebrates. Mouse and human GTRAP3–18 genes contain three exons separated by two introns. The GTRAP3–18 gene is found on mouse chromosome 6D3 and on human chromosome 3p14, a susceptibility locus for cancer and epilepsy. GTRAP3–18 protein and RNA were found both in neuronal rich regions of the brain and in non-neuronal tissues such as the kidney, heart and skeletal muscle. Mouse GTRAP3–18 inhibited EAAT3-mediated glutamate transport in a dose-dependent manner. These studies show that GTRAP3–18 is a ubiquitously expressed protein that functions as a negative regulator of EAAT3 function.