Abstract
The sodium-dependent glutamate transporter, excitatory amino acid carrier 1 (EAAC1), has been implicated in the regulation of excitatory signaling and prevention of cell death in the nervous system. There is evidence that EAAC1 constitutively cycles on and off the plasma membrane and that under steady state conditions up to 80% of the transporter is intracellular. As is observed with other neurotransmitter transporters, the activity of EAAC1 is regulated by a variety of molecules, and some of these effects are associated with redistribution of EAAC1 on and off the plasma membrane. In the present study we tested the hypothesis that a structural component of lipid rafts, caveolin-1 (Cav-1), may participate in EAAC1 trafficking. Using C6 glioma cells as a model system, co-expression of Cav-1 S80E (a dominant-negative variant) or small interfering RNA-mediated knock-down of caveolin-1 reduced cell surface expression of myc epitope-tagged EAAC1 or endogenous EAAC1, respectively. Cav-1 S80E slowed the constitutive delivery and endocytosis of myc-EAAC1. In primary cultures derived from caveolin-1 knock-out mice, a similar reduction in delivery and internalization of endogenous EAAC1 was observed. We also found that caveolin-1, caveolin-2, or Cav-1 S80E formed immunoprecipitable complexes with EAAC1 in C6 glioma and/or transfected HEK cells. Together, these data provide strong evidence that caveolin-1 contributes to the trafficking of EAAC1 on and off the plasma membrane and that these effects are associated with formation of EAAC1-caveolin complexes.
Highlights
Age-dependent neurodegeneration attributed to depletion of glutathione and consequent oxidative injury [1, 2]
A substantial amount of excitatory amino acid carrier 1 (EAAC1) immunoreactivity is observed in intracellular compartments [15,16,17], consistent with the possibility that an intracellular pool of EAAC1 is available for redistribution to the plasma membrane in the intact nervous system
The glutamate transporter EAAT2 is highly enriched in lipid rafts, and EAAT2 cell surface expression and activity are decreased by the disruption of these microdomains [25]
Summary
Materials—Sulfosuccinimidyl-2-(biotinamido) ethyl-1,3-dithiopropionate (sulfo-NHS-SS-biotin), sulfo-N-hydroxysuccinimidobiotin (sulfo-NHS-biotin), Ultralink immobilized monomeric avidin beads, and the bicinchoninic acid (BCA) protein assay reagent kit were purchased from Pierce. Measurement of EAAC1 Delivery to the Plasma Membrane— Cells were incubated in PBS containing biotin (4 ml for C6 cells and 2 ml for primary cultures at 1 mg/ml) at 37 °C as previously described [14, 36, 37]. A control plate was incubated with ice-cold biotin (4 ml for C6 cells and 2 ml for primary cultures) for 30 min to quantify the amount of biotinylated transporter at the plasma membrane at time (t) ϭ 0 min. A second plate was used to control for the efficiency of stripping These cells were not re-warmed prior to incubation with MesNa, and this sample was labeled as “0 min.”. 1 mg of cell lysate protein was incubated overnight at 4 °C with 3 g of affinity purified rabbit polyclonal anti-EAAC1 antibody (ADI). Data are presented as the mean Ϯ S.E. and compared using ANOVA with Bonferroni post hoc analysis or Student’s t test
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