title: Exon-skipping splice variants of excitatory amino acid transporter-2 (EAAT2) form heteromeric complexes with full-length EAAT2.
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.affiliation: School of Chemistry and Molecular Biosciences, University of Queensland, Brisbane 4072, Australia.
authors.forename: Florian M
authors.initials: FM
authors.lastname: Gebhardt
authors.forename: Ann D
authors.initials: AD
authors.lastname: Mitrovic
authors.forename: Daniel F
authors.initials: DF
authors.lastname: Gilbert
authors.forename: Robert J
authors.initials: RJ
authors.lastname: Vandenberg
authors.forename: Joseph W
authors.initials: JW
authors.lastname: Lynch
authors.forename: Peter R
authors.initials: PR
authors.lastname: Dodd
mesh_terms: D017398:Alternative Splicing; D001483:Base Sequence; D002460:Cell Line; D027342:Excitatory Amino Acid Transporter 2; D005091:Exons / Q000502:physiology; D050491:Glutamate Plasma Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism*; D018698:Glutamic Acid / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008564:Membrane Potentials / Q000502:physiology; D046912:Multiprotein Complexes / Q000235:genetics / Q000378:metabolism*; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D021381:Protein Transport / Q000502:physiology; D017384:Sequence Deletion
chemical_list: D027342:Excitatory Amino Acid Transporter 2; D050491:Glutamate Plasma Membrane Transport Proteins; D046912:Multiprotein Complexes; D020033:Protein Isoforms; C493397:SLC1A2 protein, human; D018698:Glutamic Acid