Elsevier

Neurochemistry International

Volume 61, Issue 4, September 2012, Pages 566-574
Neurochemistry International

The glutamate transporter, GLAST, participates in a macromolecular complex that supports glutamate metabolism

https://doi.org/10.1016/j.neuint.2012.01.013Get rights and content

Abstract

GLAST is the predominant glutamate transporter in the cerebellum and contributes substantially to glutamate transport in forebrain. This astroglial glutamate transporter quickly binds and clears synaptically released glutamate and is principally responsible for ensuring that synaptic glutamate concentrations remain low. This process is associated with a significant energetic cost. Compartmentalization of GLAST with mitochondria and proteins involved in energy metabolism could provide energetic support for glutamate transport. Therefore, we performed immunoprecipitation and co-localization experiments to determine if GLAST might co-compartmentalize with proteins involved in energy metabolism. GLAST was immunoprecipitated from rat cerebellum and subunits of the Na+/K+ ATPase, glycolytic enzymes, and mitochondrial proteins were detected. GLAST co-localized with mitochondria in cerebellar tissue. GLAST also co-localized with mitochondria in fine processes of astrocytes in organotypic hippocampal slice cultures. From these data, we hypothesized that mitochondria participate in a macromolecular complex with GLAST to support oxidative metabolism of transported glutamate. To determine the functional metabolic role of this complex, we measured CO2 production from radiolabeled glutamate in cultured astrocytes and compared it to overall glutamate uptake. Within 15 min, 9% of transported glutamate was converted to CO2. This CO2 production was blocked by inhibitors of glutamate transport and glutamate dehydrogenase, but not by an inhibitor of glutamine synthetase. Our data support a model in which GLAST exists in a macromolecular complex that allows transported glutamate to be metabolized in mitochondria to support energy production.

Introduction

Glutamate is the major excitatory neurotransmitter in the central nervous system. Synaptic glutamate must be kept at low levels (approximately 25 nM) because excessive extracellular glutamate is excitotoxic (Choi, 1992, Frandsen et al., 1989, Herman and Jahr, 2007). A family of plasma membrane glutamate transporters is responsible for clearing extracellular glutamate (Danbolt, 2001). The astrocytic glutamate transporters, GLAST, and GLT-1, are enriched in perisynaptic astrocyte processes and are responsible for the vast majority of glutamate uptake in the central nervous system (Chaudhry et al., 1995, Danbolt, 2001, Robinson, 1998, Sheldon and Robinson, 2007). GLT-1 is the predominant transporter in the forebrain, whereas GLAST is the predominant transporter in the cerebellum.

Glutamate transporters use the Na+-electrochemical gradient maintained by the Na+/K+ ATPase to drive glutamate clearance. This process is energetically costly because 3Na+ ions must be co-transported with each glutamate molecule, glutamate is transported against a steep gradient, and transport occurs in spatially restricted processes. The Na+/K+ ATPase is physically and functionally coupled to glutamate transporters (Rose et al., 2009). Our laboratory recently demonstrated that GLT-1 exists in a macromolecular complex that includes the Na+/K+ ATPase, most of the enzymes involved in glycolysis, and mitochondria (Genda et al., 2011). This work lead us to hypothesize that these proteins/organelles may exist in a complex with glutamate transporters in order to maintain the Na+-electrochemical gradient necessary to drive glutamate uptake.

Classically, it is believed that once transported into astrocytes, glutamate is converted to glutamine by glutamine synthetase (Norenberg and Martinez-Hernandez, 1979, Palmada and Centelles, 1998, Waniewski and Martin, 1986). Glutamine is then transported back into the presynaptic neuron and converted to glutamate, creating a glutamate–glutamine cycle. However, alternative metabolic pathways for glutamate are also described (Schousboe et al., 1993, Westergaard et al., 1995). Glutamate can be converted to alpha-ketoglutarate by transaminases or by glutamate dehydrogenase (Plaitakis et al., 2011). Alpha-ketoglutarate is a tricarboxylic acid (TCA) cycle intermediate involved in oxidative phosphorylation. Thus, the glutamate carbon backbone can be broken down to produce ATP and CO2. This pathway would be energetically favorable because oxidation of glutamate could potentially offset some of the energetic cost of glutamate transport. In contrast, conversion of glutamate to glutamine requires additional energy in the form of ATP.

We hypothesize that a macromolecular complex exists between GLAST, mitochondria, glycolytic enzymes, and the Na+/K+ ATPase. We performed co-immunoprecipitations to determine if mitochondrial proteins, glycolytic enzymes, and the Na+/K+ ATPase might be physically coupled to GLAST. We performed immunofluorescence experiments to determine whether GLAST co-localizes with mitochondria in vivo. We transfected astrocytes in hippocampal slice cultures with fluorescently labeled GLAST and a fluorescently labeled mitochondrial protein to determine whether co-localization of GLAST and mitochondria can occur within individual astrocytic processes. We hypothesize that this complex might exist to metabolically support glutamate transport within the spatially restricted space of these processes. To determine the metabolic role of a complex involving both GLAST and mitochondria, we adapted an assay to measure CO2 produced in cultured astrocytes.

Section snippets

Immunoprecipitation

Immunoprecipitations of GLAST were performed using Pierce crosslink immunoprecipitation columns (Thermo Scientific, Rockford, IL). Antibody (10–15 μg) was cross-linked to the column resin per manufacturer’s instructions. Cerebellar tissue was harvested from adult male Sprague–Dawley rats after euthanasia by decapitation, to avoid potential effects of anesthetic agents, and homogenized in immunoprecipitation buffer (150 mM NaCl, 1 mM EDTA, 100 mM Tris–HCl, pH 7.4, 1% Triton-X-100, and 1% sodium

Results

To determine whether GLAST interacts with proteins involved in energy metabolism, we immunoprecipitated GLAST from rat cerebellum and performed Western blot analysis for candidate interacting partners. Immunoprecipitates of GLAST from rat cerebellum contained subunits of the Na+/K+ ATPase, glycolytic enzymes, and mitochondrial proteins (Fig. 2). Specifically, we detected the α1, α2, and β1 subunits of the Na+/K+ ATPase in GLAST immunoprecipitations. The glycolytic enzymes GAPDH, and hexokinase

Discussion

It is increasingly apparent that proteins and organelles compartmentalize within cells to support specific functions (de Brito and Scorrano, 2008, Garcia-Perez et al., 2008, Sheng, 2001). We found that subunits of the Na+/K+ ATPase, glycolytic enzymes and mitochondrial proteins co-immunoprecipitate with GLAST. We also found that GLAST co-localizes with mitochondria in cerebellum and in fine processes of astrocytes in hippocampal slice cultures. To the best of our knowledge, prior to this work,

Acknowledgments

This work was supported by the Analytical Neurochemistry Core of the Institutional Intellectual and Developmental Disabilities Research Center (P30HD26979). D.E.B. was supported by an Institutional Research and Academic Career Development Award (K12GM081259). J.G.J. was partially supported by a Training Grant in Neurodevelopmental Disabilities (T32NS007413). M.M.M. was supported by the University of Pennsylvania Postbaccalaureate Research Education Program (R25GM071745). We thank the members of

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