N-Methyl-d-aspartate Receptor-dependent Regulation of the Glutamate Transporter Excitatory Amino Acid Carrier 1

Elisa A Waxman,Isabelle Baconguis,David R Lynch,Michael B Robinson

doi:10.1074/jbc.m702278200

Abstract

The neuronal transporter excitatory amino acid carrier 1 (EAAC1) is enriched in perisynaptic regions, where it may regulate synaptic spillover of glutamate. In this study we examined potential interactions between EAAC1 and ionotropic glutamate receptors. N-Methyl-D-aspartate (NMDA) receptor subunits NR1, NR2A, and NR2B, but not the alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor subunit GluR2, were co-immunoprecipitated with EAAC1 from neuron-enriched hippocampal cultures. A similar interaction was observed in C6 glioma and human embryonic kidney cells after co-transfection with Myc epitope-tagged EAAC1 and NMDA receptor subunits. Co-transfection of C6 glioma with the combination of NR1 and NR2 subunits dramatically increased (approximately 3-fold) the amount of Myc-EAAC1 that can be labeled with a membrane-impermeable biotinylating reagent. In hippocampal cultures, brief (5 min), robust (100 microM NMDA, 10 microM glycine) activation of the NMDA receptor decreased biotinylated EAAC1 to approximately 50% of control levels. This effect was inhibited by an NMDA receptor antagonist, intracellular or extracellular calcium chelators, or hypertonic sucrose. Glutamate, alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid with cyclothiazide, and thapsigargin mimicked the effects of NMDA. These studies suggest that NMDA receptors interact with EAAC1, facilitate cell surface expression of EAAC1 under basal conditions, and control internalization of EAAC1 upon activation. This NMDA receptor-dependent regulation of EAAC1 provides a novel mechanism that may shape excitatory signaling during synaptic plasticity and/or excitotoxicity.

Highlights

Interaction of excitatory amino acid carrier 1 (EAAC1) and NMDA Receptor Subunits—Because EAAC1 and ionotropic glutamate receptors may overlap in distribution [15, 17] and be regulated through similar mechanisms (Ref. 21; for a recent discussion, see Ref. 33), we examined potential interactions between EAAC1 and ionotropic glutamate receptors in primary hippocampal cultures
Because anti-EAAC1 immunoprecipitated NR1, NR2A, and NR2B, we aimed to identify the subpopulation of NMDA receptors with which EAAC1 may interact by immunoblotting for post-synaptic density 95 (PSD-95), a scaffolding protein of NMDA receptors in the postsynaptic density [40]
In the present study we identified novel physical and functional interactions between EAAC1 and NMDA receptor subunits NR1, NR2A, and NR2B in primary hippocampal cultures and C6 glioma

Summary

Introduction

In area CA1 of the hippocampus, a neuronal transporter limits synaptic spillover of glutamate and activation of NMDA receptors [9]. Because EAAC1 is the only neuronal transporter found in this location, this observation suggests that EAAC1 may control activation of some subtypes of NMDA receptors. Some studies suggest that glutamate transporters contribute to excitotoxicity through transporter reversal during insults such as ischemia [13, 14] Based on these studies, EAAC1 may play important roles in NMDA receptor-mediated excitotoxicity. LTP increases glutamate uptake activity, which has pharmacological properties consistent with EAAC1 [21] This effect is associated with an increase in EAAC1 immunoreactivity in a subcellular fraction enriched in plasma membranes and is blocked by NMDA receptor antagonists [21]. EAAC1 and ionotropic glutamate receptors may be regulated by similar mechanisms that are normally controlled by NMDA receptor stimulation

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