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.
Highlights
The amino acid sequence of an aspartate transporter from Pyrococcus horikoshii (GltPh) is 36% homologous to excitatory amino acid transporter-2 (EAAT2); many residues implicated in glutamate binding and transport are highly conserved between all Excitatory amino acid transporters (EAATs) [3, 4]
Expression of EAAT2 Splice Variants—Western blot analysis was carried out using a pan-EAAT2 primary antibody on HEK293 cells expressing EAAT2wt and EAAT2 splice variants
EAAT2⌬7 and EAAT2⌬9 showed a major band at ϳ50 –58 kDa, but only EAAT2⌬9 showed bands at high molecular mass, which suggests that EAAT2⌬9 is able to form homotrimers but that EAAT2⌬7 is unable to do so
Summary
Plasmid Construction—Plasmids encoding EAAT2 and each variant were inserted into pcDNA 3.1 mammalian expression vectors (Invitrogen). For amino-terminal tags, KpnI restriction enzyme sites were introduced at the 5Ј or 3Ј termini of CFP and YFP and ligated with KpnI-linearized EAAT2wt-pcDNA3.1 or EAAT2variant-pcDNA3.1 cDNA using a Fast-Link Kit (Epicenter). Transfection of HEK293 Cells—For Western blot analysis and imaging, 5 ϫ 105 cells/well were aliquoted in 6-well plates. 1 ϫ 105 cells were plated on coverslips in single well dishes and co-transfected with 0.25–5 g of plasmid encoding EAAT2wt or EAAT2 variants and 0.25 g of eGFP-IRES (Stratagene). Control experiments were conducted on cells transfected with 0.1 g of CFP-tagged DNA and 0.2 g of pcDNA3.1 vector DNA. Prior to imaging DMEM was aspirated and cells were washed twice with Leibovitz’s L-15 medium (Invitrogen). Statistical significance was determined by paired Student’s t tests, or by analysis of variance with appropriate post hoc tests where indicated
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