ORIGINAL PAPER Distribution of Glutamate Transporter GLAST in Membranes of Cultured Astrocytes in the Presence of Glutamate Transport Substrates and ATP Jae-Won Shin Æ Khoa T. D. Nguyen Æ David V. Pow Æ Toby Knight Æ Vlado Buljan Æ Maxwell R. Bennett Æ Vladimir J. Balcar Accepted: 20 April 2009 / Published online: 8 May 2009 Ó Springer Science+Business Media, LLC 2009 Abstract Neurotransmitter L-glutamate released at cen- tral synapses is taken up and ‘‘recycled’’ by astrocytes using glutamate transporter molecules such as GLAST and GLT. Glutamate transport is essential for prevention of glutamate neurotoxicity, it is a key regulator of neuro- transmitter metabolism and may contribute to mechanisms through which neurons and glia communicate with each other. Using immunocytochemistry and image analysis we have found that extracellular D-aspartate (a typical sub- strate for glutamate transport) can cause redistribution of GLAST from cytoplasm to the cell membrane. The process appears to involve phosphorylation/dephosphorylation and requires intact cytoskeleton. Glutamate transport ligands L-trans-pyrrolidine-2,4-dicarboxylate and DL-threo-3-ben- zyloxyaspartate but not anti,endo-3,4-methanopyrrolidine dicarboxylate have produced similar redistribution of GLAST. Several representative ligands for glutamate receptors whether of ionotropic or metabotropic type, were found to have no effect. In addition, extracellular ATP induced formation of GLAST clusters in the cell mem- branes by a process apparently mediated by P2 receptors. The present data suggest that GLAST can rapidly and specifically respond to changes in the cellular environment thus potentially helping to fine-tune the functions of astrocytes. Keywords Astrocytes Á Glutamate synapses Á Glutamate transport Á GLAST Á GLT Á Brain metabolism Á Na ? , K ? -dependent ATPase Á Neurotransmitter metabolism Á Regulation of transporters Á Phosphorylation and dephosphorylation of membrane proteins Introduction Acidic amino acids L-aspartate (L-asp) and L-glutamate (L-glu) act on a set of membrane-located receptors that function as ion-gated channels, changing membrane per- meability to Na ? and Ca 2? (‘‘ionotropic glutamate recep- tors’’, iGluR) and depolarizing (‘‘exciting’’) the neurons in the central nervous system. In addition, L-glu, but not L-asp, acts on distinct G-protein linked receptors (‘‘metabotropic glutamate receptors’’, mGluR) that activate intracellular signalling pathways [1]. Thus L-glu and L-asp, can act as signalling agents in the intercellular communication and, indeed, L-glu appears to be the major neurotransmitter at the excitatory synapses in brain (reviews [2, 3]). L-Glu is stored in presynaptic vesicles and is released from synaptic nerve endings by depolarization-coupled, Ca 2? -dependent mechanisms. Active Na ? /K ? -dependent transport of L-glu (GluT) provides a mechanism that reg- ulates concentrations of L-glu at the synapse, preventing excessive stimulation of glutamate receptors that could be harmful and cause pathological neurodegeneration [4]. The authors J.-W. Shin and K. T. D. Nguyen have contributed equally. J.-W. Shin Á K. T. D. Nguyen Á T. Knight Á V. J. Balcar (&) Anatomy and Histology, School of Medical Sciences and Bosch Institute for Biomedical Research, The University of Sydney, Sydney, NSW 2006, Australia e-mail: vibar@anatomy.usyd.edu.au D. V. Pow Centre for Clinical Research, Royal Brisbane and Womens Hospital, The University of Queensland, Brisbane, QLD 4029, Australia V. Buljan Á M. R. Bennett Brain and Mind Research Institute, The University of Sydney, Sydney, NSW 2006, Australia 123 Neurochem Res (2009) 34:1758–1766 DOI 10.1007/s11064-009-9982-z