Serine‐O‐sulphate transport by the human glutamate transporter, EAAT2

Vandenberg, Robert J.; Mitrovic, Ann D.; Johnston, Graham A.R.

doi:10.1038/sj.bjp.0701776

Cited by 33 publications

(28 citation statements)

References 24 publications

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“…This Gly for Ser difference has previously been investigated using the rat equivalent of human EAAT2 (19), and it was found that the S440G mutation reduced the affinity of the EAAT2-selective blocker, dihydrokainate, and also allowed Li ϩ to drive transport. Two previous studies have also demonstrated that the affinity of Na ϩ for the transporter is dependent upon the substrate being transported (20,21), which provided hints of a link between substrate/blocker interactions and Na ϩ binding. These observations provide us with the tools to investigate the specificity of the coupling process and why compounds such as MPDC and 4MG are very good substrates for EAAT1 and are either blockers or poor substrates of A higher L-glutamate concentration than MPDC and 4MG was used because of its higher EC 50 in Li ϩ (see Table 1).…”

Section: Discussionmentioning

confidence: 99%

The Role of Cation Binding in Determining Substrate Selectivity of Glutamate Transporters

Huang¹,

Ryan²,

Vandenberg

2009

Journal of Biological Chemistry

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Glutamate transport is coupled to the co-transport of 3Na ؉ and 1H؉ and the countertransport of 1 K ؉ . However, the mechanism of how this process occurs is not well understood. The crystal structure of an archaeal homolog of the human glutamate transporters, Glt Ph , has provided the framework to begin to understand the mechanism of transport. The glutamate transporter EAAT2 is different from other subtypes in two respects. First, Li ؉ cannot support transport by EAAT2, whereas it can support transport by the other excitatory amino acid transporters, and second, EAAT2 is sensitive to a wider range of blockers than other subtypes. We have investigated the relationship between the cation driving transport and whether the glutamate analogues, L-anti-endo-3,4-methanopyrrolidinedicarboxylic acid (MPDC) and (2S,4R)-4-methylglutamate (4MG), are substrates or blockers of transport. We have also investigated the molecular basis for these differences. EAAT2 has a Ser residue at position 441 with hairpin loop 2, whereas the corresponding residue in EAAT1 is a Gly residue. We demonstrate that if the transporter has a Ser residue at this position, then 4MG and MPDC are poor substrates in Na ؉ , and Li ؉ cannot support transport of any substrate. Conversely, if the transporter has a Gly residue at this position, then in Na ؉ 4MG and MPDC are substrates with efficacy comparable with glutamate, but in Li ؉ 4MG and MPDC are poor substrates relative to glutamate. This Ser/Gly residue is located between the bound substrate and one of the cation binding sites, which provides an explanation for the coupling of substrate and cation binding.

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Section: Discussionmentioning

confidence: 99%

The Role of Cation Binding in Determining Substrate Selectivity of Glutamate Transporters

Huang¹,

Ryan²,

Vandenberg

2009

Journal of Biological Chemistry

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“…The first molecular probe, L-serine-O-sulfate, is a substrate for transporters which displays a difference in affinity (8,16,23). Chloride permeability, the second molecular probe, is markedly different between EAAT1 transport of D-aspartate and EAAT2 transport of D-aspartate (17) and was employed to delineate further the differences in EAAT1 and EAAT2 related to the pore of the transporter.…”

Section: Discussionmentioning

confidence: 99%

“…L-Serine-O-sulfate is a substrate for both EAAT1 and EAAT2, but it is a less potent substrate on EAAT2 than EAAT1. In addition, the EC 50 2 values for L-serine-O-sulfate transport by EAAT2 are steeply voltage-dependent but relatively constant for transport by EAAT1 (16). This difference between EAAT1 and EAAT2 in L-serine-O-sulfate transport is likely to reflect a difference in the structure of the pore of the two transporters.…”

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confidence: 95%

Identification of Functional Domains of the Human Glutamate Transporters EAAT1 and EAAT2

Mitrovic¹,

Amara²,

Johnston³

et al. 1998

Journal of Biological Chemistry

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Glutamate transporters serve the important function of mediating removal of glutamate released at excitatory synapses and maintaining extracellular concentrations below excitotoxic levels. Excitatory amino acid transporter subtypes EAAT1 and EAAT2 have a high degree of sequence homology and similar predicted topology and yet display a number of functional differences. Several recombinant chimeric transporters were generated to identify domains that contribute to functional differences between EAAT1 and EAAT2. Wild-type transporters and chimeric transporters were expressed in Xenopus laevis oocytes, and electrogenic transport was studied under voltage clamp conditions. The differential sensitivity of EAAT1 and EAAT2 to transport blockers, kainate, threo-3-methylglutamate, and (2S,4R)-4-methylglutamate as well as L-serine-O-sulfate transport and chloride permeability were employed to characterize chimeric transporters. One particular region, transmembrane domains 9 and 10, plays an important role in defining these functional differences. The intracellular carboxyl-terminal region may also play a minor role in conferring an effect on chloride permeability. This study provides important insight into the identification of functional domains that determine differences among glutamate transporter subtypes.L-Glutamate transport in the central nervous system and also in peripheral organs is mediated by a family of structurally related membrane proteins. In the central nervous system, glutamate transporters play an important role by mediating removal of glutamate released at excitatory synapses and also influencing the kinetics of glutamate receptor activation (1, 2). Complementary DNAs encoding five different glutamate transporters have been identified (3-7). The human clones have been named Excitatory Amino Acid Transporters 1-5 (EAAT1-5) (6-8). The recent cloning and expression of recombinant glutamate transporter proteins in heterologous expression systems allow a number of questions concerning the molecular basis for transporter function to be addressed. In particular, an important aspect of such studies is the identification of domains involved in substrate recognition and substrate translocation through the membrane. In this study we have constructed a series of chimeric glutamate transporters, using the EAAT1 and EAAT2 subtypes, and exploited the pharmacological and electrophysiological differences between the two subtypes to map functional domains of these proteins.To make meaningful predictions about the functional role of various domains, an understanding of the structure of the transporters is particularly useful. The predicted amino acid sequences of EAAT1 and EAAT2 are 65% identical, and if conservative substitutions are allowed the degree of relatedness is increased to 80% (8). These two proteins are likely to form very similar structures and therefore are good candidates for the generation of functional chimeras. Through the use of hydrophobicity analysis of the amino acid sequences it has been predicted t...

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“…Moreover, L-serine-O-sulfate, L-homocysteate, L-homocysteine sulfinate, L-ibotenate, and L-quisqualate activate metabotropic glutamate receptors (26,119,184,248), and (S)-4-CPG is a Group I metabotropic glutamate receptor antagonist (22). L-serine-O-sulfate also acts as an inhibitor of serine racemase (199) and aspartate aminotransferase (273) as well as an EAAT substrate (278). While Lquisqualate and (S)-4-CPG are the most potent inhibitors of system x c --mediated glutamate uptake, they are less well transported by system x c -than L-ibotenate and noncyclic glutamate analogs.…”

Section: Fig 2 System X Cmentioning

confidence: 99%

The Cystine/Glutamate Antiporter System x_c⁻in Health and Disease: From Molecular Mechanisms to Novel Therapeutic Opportunities

Lewerenz

Hewett

Huang

et al. 2013

Antioxidants & Redox Signaling

756

655

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The antiporter system x c -imports the amino acid cystine, the oxidized form of cysteine, into cells with a 1:1 counter-transport of glutamate. It is composed of a light chain, xCT, and a heavy chain, 4F2 heavy chain (4F2hc), and, thus, belongs to the family of heterodimeric amino acid transporters. Cysteine is the rate-limiting substrate for the important antioxidant glutathione (GSH) and, along with cystine, it also forms a key redox couple on its own. Glutamate is a major neurotransmitter in the central nervous system (CNS). By phylogenetic analysis, we show that system x c -is a rather evolutionarily new amino acid transport system. In addition, we summarize the current knowledge regarding the molecular mechanisms that regulate system x c -, including the transcriptional regulation of the xCT light chain, posttranscriptional mechanisms, and pharmacological inhibitors of system x c -. Moreover, the roles of system x c -in regulating GSH levels, the redox state of the extracellular cystine/cysteine redox couple, and extracellular glutamate levels are discussed. In vitro, glutamate-mediated system x c -inhibition leads to neuronal cell death, a paradigm called oxidative glutamate toxicity, which has successfully been used to identify neuroprotective compounds. In vivo, xCT has a rather restricted expression pattern with the highest levels in the CNS and parts of the immune system. System x c -is also present in the eye. Moreover, an elevated expression of xCT has been reported in cancer. We highlight the diverse roles of system x c -in the regulation of the immune response, in various aspects of cancer and in the eye and the CNS. Antioxid. Redox Signal. 18, 522-555.

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Serine‐O‐sulphate transport by the human glutamate transporter, EAAT2

Cited by 33 publications

References 24 publications

The Role of Cation Binding in Determining Substrate Selectivity of Glutamate Transporters

The Role of Cation Binding in Determining Substrate Selectivity of Glutamate Transporters

Identification of Functional Domains of the Human Glutamate Transporters EAAT1 and EAAT2

The Cystine/Glutamate Antiporter System x_c⁻in Health and Disease: From Molecular Mechanisms to Novel Therapeutic Opportunities

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Serine‐O‐sulphate transport by the human glutamate transporter, EAAT2

Cited by 33 publications

References 24 publications

The Role of Cation Binding in Determining Substrate Selectivity of Glutamate Transporters

The Role of Cation Binding in Determining Substrate Selectivity of Glutamate Transporters

Identification of Functional Domains of the Human Glutamate Transporters EAAT1 and EAAT2

The Cystine/Glutamate Antiporter System xc−in Health and Disease: From Molecular Mechanisms to Novel Therapeutic Opportunities

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The Cystine/Glutamate Antiporter System x_c⁻in Health and Disease: From Molecular Mechanisms to Novel Therapeutic Opportunities