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

Gebhardt, FM, Mitrovic, AD, Gilbert, DF, Vandenberg, RJ, Lynch, JW and Dodd, PR (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: 31313-31324. doi:10.1074/jbc.M110.153494

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Author Gebhardt, FM
Mitrovic, AD
Gilbert, DF
Vandenberg, RJ
Lynch, JW
Dodd, PR
Title Exon-skipping splice variants of excitatory amino acid transporter-2 (EAAT2) form heteromeric complexes with full-length EAAT2
Journal name Journal of Biological Chemistry   Check publisher's open access policy
ISSN 0021-9258
1083-351X
Publication date 2010-10-08
Sub-type Article (original research)
DOI 10.1074/jbc.M110.153494
Open Access Status File (Publisher version)
Volume 285
Issue 41
Start page 31313
End page 31324
Total pages 12
Place of publication Bethesda, MD, United States
Publisher American Society for Biochemistry and Molecular Biology
Collection year 2011
Language eng
Formatted 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 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 EC50 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.
© 2010 by The American Society for Biochemistry and Molecular Biology, Inc.
Keyword Amyotrophic-lateral-sclerosis
Dependent asparate transporter
Neuronal glutamate transporter
Functional-characterization
Q-Index Code C1
Q-Index Status Confirmed Code
Institutional Status UQ

Document type: Journal Article
Sub-type: Article (original research)
Collections: Official 2011 Collection
School of Chemistry and Molecular Biosciences
 
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Created: Sun, 28 Nov 2010, 00:04:15 EST