title: Displacing hexokinase from mitochondrial voltage-dependent anion channel impairs GLT-1-mediated glutamate uptake but does not disrupt interactions between GLT-1 and mitochondrial proteins.
abstract: The glutamate transporter GLT-1 is the major route for the clearance of extracellular glutamate in the forebrain, and most GLT-1 protein is found in astrocytes. This protein is coupled to the Na(+) electrochemical gradient, supporting the active intracellular accumulation of glutamate. We recently used a proteomic approach to identify proteins that may interact with GLT-1 in rat cortex, including the Na(+)/K(+) -ATPase, most glycolytic enzymes, and several mitochondrial proteins. We also showed that most GLT-1 puncta (∼ 70%) are overlapped by mitochondria in astroglial processes in organotypic slices. From this analysis, we proposed that the glycolytic enzyme hexokinase (HK)-1 might physically form a scaffold to link GLT-1 and mitochondria because HK1 is known to interact with the outer mitochondrial membrane protein voltage-dependent anion channel (VDAC). The current study validates the interactions among HK-1, VDAC, and GLT-1 by using forward and reverse immunoprecipitations and provides evidence that a subfraction of HK1 colocalizes with GLT-1 in vivo. A peptide known to disrupt the interaction between HK and VDAC did not disrupt interactions between GLT-1 and several mitochondrial proteins. In parallel experiments, displacement of HK from VDAC reduced GLT-1-mediated glutamate uptake. These results suggest that, although HK1 forms coimmunoprecipitatable complexes with both VDAC and GLT-1, it does not physically link GLT-1 to mitochondrial proteins. However, the interaction of HK1 with VDAC supports GLT-1-mediated transport activity.
authors.affiliation: Children's Hospital of Philadelphia Research Institute, Philadelphia, Pennsylvania.|Department of Pediatrics, University of Pennsylvania, Philadelphia, Pennsylvania.
authors.forename: Joshua G
authors.initials: JG
authors.lastname: Jackson
authors.affiliation: Children's Hospital of Philadelphia Research Institute, Philadelphia, Pennsylvania.|Department of Pharmacology, University of Pennsylvania, Philadelphia, Pennsylvania.
authors.forename: John C
authors.initials: JC
authors.lastname: O'Donnell
authors.affiliation: Children's Hospital of Philadelphia Research Institute, Philadelphia, Pennsylvania.
authors.forename: Elizabeth
authors.initials: E
authors.lastname: Krizman
authors.affiliation: Children's Hospital of Philadelphia Research Institute, Philadelphia, Pennsylvania.|Department of Pediatrics, University of Pennsylvania, Philadelphia, Pennsylvania.|Department of Pharmacology, University of Pennsylvania, Philadelphia, Pennsylvania.
authors.forename: Michael B
authors.initials: MB
authors.lastname: Robinson
mesh_terms: D000818:Animals; D002540:Cerebral Cortex / Q000378:metabolism* / Q000648:ultrastructure; D027342:Excitatory Amino Acid Transporter 2 / Q000378:metabolism*; D018698:Glutamic Acid / Q000378:metabolism*; D006593:Hexokinase / Q000737:chemistry / Q000378:metabolism*; D047468:Immunoprecipitation; D008297:Male; D051336:Mitochondrial Membranes / Q000378:metabolism; D024101:Mitochondrial Proteins / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D013574:Synaptosomes / Q000378:metabolism; D050994:Voltage-Dependent Anion Channels / Q000378:metabolism*
chemical_list: D027342:Excitatory Amino Acid Transporter 2; D024101:Mitochondrial Proteins; C493395:Slc1a2 protein, rat; D050994:Voltage-Dependent Anion Channels; D018698:Glutamic Acid; D006593:Hexokinase
keywords: glutamate transporter; hexokinase; mitochondria