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Identification of components of a glutamate homeostasis complex in astrocytes

P. Poronnik,2 A. Lee1 and D.V. Pow,1 1UQCCR and Perinatal Research Centre, The University of Queensland, QLD 4072, Australia and 2School of Medical Sciences, RMIT University, VIC 3083, Australia.

Efficient glutamate transport into astrocytes is essential for termination of glutamatergic neurotransmission. Uptake by proteins such as GLAST (EAAT1) requires a sodium gradient, formed by the actions of a sodium-potassium ATPase (NKA), and the rapid catabolism of the accumulated glutamate by glutamine synythetase (GS). We demonstrate, by co-immunoprecipitation experiments (n=12) that GLAST associates with NKA α 1 in the rat brain. GST-pulldown assays demonstrate that the cytoplasmic loop region (residues 350 775) of NKA α 1 pulls down GLAST from brain lysate. Similarly, the extreme C-terminal tail (residues 513 -543) of GLAST pulls down NKA α 1 from brain lysate. Confocal microscopy studies revealed that GLAST co-localised with NKA α 1. We have examined D-aspartate uptake in transfected Cos-7 cells to study the effects of co-expression of GLAST and a construct encoding the cytoplasmic loop region (residues 350 775) of NKA α 1. The construct was predicted to block normal interactions between GLAST and NKA α 1. Co-expression led to a significant decrease (∼20%) in D-aspartate uptake when compared to the control. The interaction between the cytoplasmic loop region of NKA α 1 and the C-terminal tail of GLAST is likely to involve other accessory proteins. Similarly we demonstrate by immunoprecipitation studies that the enzyme GS is part of this multi-molecular complex. We have previously demonstrated that NHERF1 anchors GLAST to the cytoskeleton, and confirm here that NHERF1 is part of the complex containing GS, GLAST and NKA α 1. These findings indicate that multiple protein interactions may be required for efficient glutamate transport and that these interactions may represent novel pharmacological targets in conditions such as stroke.