The Four Major N- and C-Terminal Splice Variants of the Excitatory Amino Acid Transporter GLT-1 Form Cell Surface Homomeric and Heteromeric Assemblies

Peacey, Eleanor; Miller, Christopher C.J.; Dunlop, John; Rattray, Marcus

doi:10.1124/mol.108.052829

Cited by 43 publications

(54 citation statements)

References 45 publications

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“…We have a particular interest in expression and regulation of the glutamate transporter, GLT-1 [27,44] that is responsible for approximately 95% of all L-glutamate uptake in the CNS and accounts for about 1% of all CNS protein [5,45,46]. Recent transcriptomic data, confirms that GLT-1 is predominantly enriched in astrocytes [14,45,47].…”

Section: Discussionmentioning

confidence: 86%

Astrocytes Grown in Alvetex® Three Dimensional Scaffolds Retain a Non-reactive Phenotype

2016

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Protocols which permit the extraction of primary astrocytes from either embryonic or postnatal mice are well established however astrocytes in culture are different to those in the mature CNS. Three dimensional (3D) cultures, using a variety of scaffolds may enable better phenotypic properties to be developed in culture. We present data from embryonic (E15) and postnatal (P4) murine primary cortical astrocytes grown on coated coverslips or a 3D polystyrene scaffold, Alvetex. Growth of both embryonic and postnatal primary astrocytes in the 3D scaffold changed astrocyte morphology to a mature, protoplasmic phenotype. Embryonic-derived astrocytes in 3D expressed markers of mature astrocytes, namely the glutamate transporter GLT-1 with low levels of the chondroitin sulphate proteoglycans, NG2 and SMC3. Embroynic astrocytes derived in 3D show lower levels of markers of reactive astrocytes, namely GFAP and mRNA levels of LCN2, PTX3, Serpina3n and Cx43. Postnatal-derived astrocytes show few protein changes between 2D and 3D conditions. Our data shows that Alvetex is a suitable scaffold for growth of astrocytes, and with appropriate choice of cells allows the maintenance of astrocytes with the properties of mature cells and a non-reactive phenotype.

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Section: Discussionmentioning

confidence: 86%

Astrocytes Grown in Alvetex® Three Dimensional Scaffolds Retain a Non-reactive Phenotype

2016

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“…Therefore, our observation that GLT-1a is expressed in some of the same cells as the ␣3 subunit provides additional evidence that, in addition to the well established expression in astrocytes, GLT-1a is also expressed in some neurons. Indeed, at least four splice variants of GLT-1 are expressed in rodent brain (Peacey et al, 2009), and a growing number of reports have demonstrated neuronal expression of GLT-1 (Chen et al, 2004;Bassan et al, 2008;González-González et al, 2008).…”

Section: Discussionmentioning

confidence: 99%

Glutamate Transporter Coupling to Na,K-ATPase

Rose

Koo²,

Antflick³

et al. 2009

Journal of Neuroscience

279

232

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Deactivation of glutamatergic signaling in the brain is mediated by glutamate uptake into glia and neurons by glutamate transporters. Glutamate transporters are sodium-dependent proteins that putatively rely indirectly on Na,K-ATPases to generate ion gradients that drive transmitter uptake. Based on anatomical colocalization, mutual sodium dependency, and the inhibitory effects of the Na,K-ATPase inhibitor ouabain on glutamate transporter activity, we postulated that glutamate transporters are directly coupled to Na,K-ATPase and that Na,K-ATPase is an essential modulator of glutamate uptake. Na,K-ATPase was purified from rat cerebellum by tandem anion exchange and ouabain affinity chromatography, and the cohort of associated proteins was characterized by mass spectrometry. The ␣1-␣3 subunits of Na,K-ATPase were detected, as were the glutamate transporters GLAST and GLT-1, demonstrating that glutamate transporters copurify with Na,K-ATPases. The link between glutamate transporters and Na,K-ATPase was further established by coimmunoprecipitation and colocalization. Analysis of the regulation of glutamate transporter and Na,K-ATPase activities was assessed using

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“…FRET was used to show that EAAT2wt not only forms heteromeric complexes with EAAT2b, as has been described before (18), but also with EAAT2⌬7 and EAAT⌬9. For EAAT2wt:EAAT2⌬7 or EAAT2wt:EAAT2⌬9 heteromers, FRET was dependent on the position of the fluorescent tags.…”

Section: Discussionmentioning

confidence: 99%

“…EAAT2b and EAAT2wt can also form heteromeric complexes (see above and Ref. 18). EAAT2⌬7 and EAAT2⌬9 were co-transfected in various ratios with EAAT2b.…”

Section: Expression Of Eaat2 Splicementioning

confidence: 99%

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Exon-skipping Splice Variants of Excitatory Amino Acid Transporter-2 (EAAT2) Form Heteromeric Complexes with Full-length EAAT2

Gebhardt

Mitrovic

Gilbert

et al. 2010

Journal of Biological Chemistry

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The glial transporter excitatory amino acid transporter-2 (EAAT2) is the main mediator of glutamate clearance in brain. The wild-type transporter (EAAT2wt) forms trimeric membrane complexes in which each protomer functions autonomously. Several EAAT2 variants are found in control and Alzheimer-diseased human brains; their expression increases with pathological severity. These variants might alter EAAT2wt-mediated transport by abrogating membrane trafficking, or by changing the configuration or functionality of the assembled transporter complex. HEK293 cells were transfected with EAAT2wt; EAAT2b, a C-terminal variant; or either of two exonskipping variants: alone or in combination. Surface biotinylation studies showed that only the exon-7 deletion variant was not trafficked to the membrane when transfected alone, and that all variants could reach the membrane when co-transfected with EAAT2wt. Fluorescence resonance energy transfer (FRET) studies showed that co-transfected EAAT2wt and EAAT2 splice variants were expressed in close proximity. Glutamate transporter function was measured using a whole cell patch clamp technique, or by changes in membrane potential indexed by a voltage-sensitive fluorescent dye (FMP assay): the two methods gave comparable results. Cells transfected with EAAT2wt or EAAT2b showed glutamate-dependent membrane potential changes consistent with functional expression. Cells transfected with EAAT2 exon-skipping variants alone gave no response to glutamate. Co-transfection of EAAT2wt (or EAAT2b) and splice variants in various ratios significantly raised glutamate EC 50 and decreased Hill coefficients. We conclude that exon-skipping variants form heteromeric complexes with EAAT2wt or EAAT2b that traffic to the membrane but show reduced glutamate-dependent activity. This could allow glutamate to accumulate extracellularly and promote excitotoxicity. Excitatory amino acid transporters (EAATs)6 are high-affinity, sodium-dependent glutamate carriers from solute carrier family 1 (SLC1). Five EAATs have been cloned from animal and human tissue. EAAT1 (SLC1A3; GLAST in rodents) and EAAT2 (SLC1A2; GLT1) are primarily expressed in astroglia (1). Overall, EAAT2 is responsible for most of the glutamate transport in the adult brain (1, 2).The amino acid sequence of an aspartate transporter from Pyrococcus horikoshii (Glt Ph ) is 36% homologous to EAAT2; many residues implicated in glutamate binding and transport are highly conserved (up to 90%) between all EAATs (3, 4). The crystal structure of Glt Ph is a trimer in which each protomer comprises eight ␣-helical transmembrane domains and two helical hairpins (5). It has been proposed that transmembrane domains, TM3, -6, -7, and -8, together with the two helical hairpins, HP1 and HP2, are essential for substrate translocation from the extracellular side into the cell. The first transmembrane segments, TM1, -2, -4, and -5, form the trimerization domain and provide stability to counterbalance the movements of the transport domains (3, 6, 7).In addition to EAAT...

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The Four Major N- and C-Terminal Splice Variants of the Excitatory Amino Acid Transporter GLT-1 Form Cell Surface Homomeric and Heteromeric Assemblies

Cited by 43 publications

References 45 publications

Astrocytes Grown in Alvetex® Three Dimensional Scaffolds Retain a Non-reactive Phenotype

Astrocytes Grown in Alvetex® Three Dimensional Scaffolds Retain a Non-reactive Phenotype

Glutamate Transporter Coupling to Na,K-ATPase

Exon-skipping Splice Variants of Excitatory Amino Acid Transporter-2 (EAAT2) Form Heteromeric Complexes with Full-length EAAT2

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