Exon-skipping splice variants of excitatory amino acid transporter-2 (EAAT2) form heteromeric complexes with full-length EAAT2.

Gebhardt F, Mitrovic A, Gilbert D, Vandenberg R, Lynch J, Dodd P (2010)

Publication Status: Published

Publication Type: Journal article, Original article

Publication year: 2010

Journal

Journal of Biological Chemistry American Society for Biochemistry and Molecular Biology

Publisher: American Society for Biochemistry and Molecular Biology

Book Volume: 285

Pages Range: 31313-24

Journal Issue: 41

DOI: 10.1074/jbc.M110.153494

Abstract

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 exon-skipping 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.

Authors with CRIS profile

Daniel Gilbert Lehrstuhl für Medizinische Biotechnologie

Involved external institutions

Queensland University of Technology (QUT) AU Australia (AU) University of Sydney (USYD) AU Australia (AU) University of Queensland AU Australia (AU)

How to cite

APA:

Gebhardt, F., Mitrovic, A., Gilbert, D., Vandenberg, R., Lynch, J., & Dodd, P. (2010). Exon-skipping splice variants of excitatory amino acid transporter-2 (EAAT2) form heteromeric complexes with full-length EAAT2. Journal of Biological Chemistry, 285(41), 31313-24. https://doi.org/10.1074/jbc.M110.153494

MLA:

Gebhardt, FM, et al. "Exon-skipping splice variants of excitatory amino acid transporter-2 (EAAT2) form heteromeric complexes with full-length EAAT2." Journal of Biological Chemistry 285.41 (2010): 31313-24.

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