361 Original Paper Cell Physiol Biochem 2009;24:361-368 Accepted: August 17, 2009 Cellular Physiology Cellular Physiology Cellular Physiology Cellular Physiology Cellular Physiology and Biochemistr and Biochemistr and Biochemistr and Biochemistr and Biochemistry Copyright © 2009 S. Karger AG, Basel Fax +41 61 306 12 34 E-Mail karger@karger.ch www.karger.com © 2009 S. Karger AG, Basel 1015-8987/09/0246-0361$26.00/0 Accessible online at: www.karger.com/cpb Regulation of the Glutamate Transporter EAAT2 by PIKfyve Eva-Maria Gehring 1 , Agathe Zürn 1 , Fabian Klaus 1 , Joerg Laufer 1 , Mentor Sopjani 1 , Ricco Lindner 1 , Nathalie Strutz-Seebohm 1,2 , Jeremy M. Tavaré 3 , Christoph Boehmer 1,4 , Monica Palmada 1,4 , Undine E. Lang 5 , Guiscard Seebohm 1,2 and Florian Lang 1 1 Department of Physiology I, University of Tübingen, 2 Department of Biochemistry I, Ruhr-University Bochum, Bochum, 3 Department of Biochemistry, School of Medical Sciences, University of Bristol, University Walk, Bristol, 4 Department of Molecular Biology, University of Essen, 5 Department of Psychiatry and Psychotherapy, Charité University Medicine Berlin, Campus Mitte, Berlin Prof. Dr. Florian Lang Physiologisches Institut I, Universität Tübingen Gmelinstr. 5, D-72076 Tübingen (Germany) Tel. +49 7071 2972194, Fax +49 7071 295618, E-Mail florian.lang@uni-tuebingen.de Key Words PIP5K3  Astrocytes  Neuroexcitability  lucocorticoids  Neurotransmission  Excitatory amino acid transporter 2 Abstract The Na + ,glutamate cotransporter EAAT2 is expressed in astrocytes and clears glutamate from the synaptic cleft. EAAT2 dependent currrent is stimulated by the serum and glucocorticoid inducible kinase SGK1. Phosphorylation targets of SGK1 include the human phosphatidylinositol-3-phosphate-5-kinase PIKfyve (PIP5K3). Nothing is known, however, on the role of PIKfyve in the regulation of EAAT2. The present experiments thus explored, whether PIKfyve expression modifies EAAT2 dependent currrent and protein abundance in the cell membrane. In Xenopus oocytes expressing EAAT2 but not in water injected oocytes application of glutamate (2 mM) induced an inward current (I glu ). Coexpression of either, SGK1 or PIKfyve, significantly enhanced I glu in EAAT2 expressing oocytes. I glu was significantly higher in Xenopus oocytes coexpressing EAAT2, SGK1 and PIKfyve than in Xenopus oocytes expressing EAAT2 and either, SGK1 or PIKfyve, alone. Additional coexpression of the inactive mutant of the serum and glucocorticoid inducible kinase K127N SGK1 did not significantly alter I glu in EAAT2 expressing oocytes and significantly decreased I glu in oocytes coexpressing EAAT2 together with PIKfyve. The stimulating effect of PIKfyve on I glu was abrogated by replacement of the serine in the SGK consensus sequence by alanine ( S318A PIKfyve). Furthermore, additional coexpression of S318A PIKfyve virtually abolished I glu in Xenopus oocytes coexpressing SGK1 and EAAT2. Confocal microscopy reveals that PIKfyve enhances the EAAT2 protein abundance in the cell membrane. The observations disclose that PIKfyve indeed participates in the regulation of EAAT2. Introduction The Na + ,glutamate cotransporter EAAT2 is primarily expressed in astrocytes [1] and accomplishes glutamate reuptake from the synaptic cleft [2]. Upregulation of EAAT2 activity provides neuroprotection [3] and impaired expression or activity of EAAT2 leads to extracellular G