title: Down-Regulation of Excitatory Amino Acid Transporters EAAT1 and EAAT2 by the Kinases SPAK and OSR1.
abstract: SPAK (SPS1-related proline/alanine-rich kinase) and OSR1 (oxidative stress-responsive kinase 1) are cell volume-sensitive kinases regulated by WNK (with-no-K[Lys]) kinases. SPAK/OSR1 regulate several channels and carriers. SPAK/OSR1 sensitive functions include neuronal excitability. Orchestration of neuronal excitation involves the excitatory glutamate transporters EAAT1 and EAAT2. Sensitivity of those carriers to SPAK/OSR1 has never been shown. The present study thus explored whether SPAK and/or OSR1 contribute to the regulation of EAAT1 and/or EAAT2. To this end, cRNA encoding EAAT1 or EAAT2 was injected into Xenopus oocytes without or with additional injection of cRNA encoding wild-type SPAK or wild-type OSR1, constitutively active (T233E)SPAK, WNK insensitive (T233A)SPAK, catalytically inactive (D212A)SPAK, constitutively active (T185E)OSR1, WNK insensitive (T185A)OSR1 or catalytically inactive (D164A)OSR1. The glutamate (2 mM)-induced inward current (I Glu) was taken as a measure of glutamate transport. As a result, I Glu was observed in EAAT1- and in EAAT2-expressing oocytes but not in water-injected oocytes, and was significantly decreased by coexpression of SPAK and OSR1. As shown for EAAT2, SPAK, and OSR1 decreased significantly the maximal transport rate but significantly enhanced the affinity of the carrier. The effect of wild-type SPAK/OSR1 on EAAT1 and EAAT2 was mimicked by (T233E)SPAK and (T185E)OSR1, but not by (T233A)SPAK, (D212A)SPAK, (T185A)OSR1, or (D164A)OSR1. Coexpression of either SPAK or OSR1 decreased the EAAT2 protein abundance in the cell membrane of EAAT2-expressing oocytes. In conclusion, SPAK and OSR1 are powerful negative regulators of the excitatory glutamate transporters EAAT1 and EAAT2.
authors.affiliation: Department of Physiology, University of Tübingen, Gmelinstr. 5, 72076, Tübingen, Germany.
authors.forename: Abeer
authors.initials: A
authors.lastname: Abousaab
authors.affiliation: Department of Physiology, University of Tübingen, Gmelinstr. 5, 72076, Tübingen, Germany.
authors.forename: Jamshed
authors.initials: J
authors.lastname: Warsi
authors.affiliation: Department of Physiology, University of Tübingen, Gmelinstr. 5, 72076, Tübingen, Germany.
authors.forename: Bernat
authors.initials: B
authors.lastname: Elvira
authors.affiliation: Department of Physiology, University of Tübingen, Gmelinstr. 5, 72076, Tübingen, Germany.
authors.forename: Ioana
authors.initials: I
authors.lastname: Alesutan
authors.affiliation: Department of Physiology, University of Tübingen, Gmelinstr. 5, 72076, Tübingen, Germany.
authors.forename: Zohreh
authors.initials: Z
authors.lastname: Hoseinzadeh
authors.affiliation: Department of Physiology, University of Tübingen, Gmelinstr. 5, 72076, Tübingen, Germany. florian.lang@uni-tuebingen.de.
authors.forename: Florian
authors.initials: F
authors.lastname: Lang
mesh_terms: D000818:Animals; D001692:Biological Transport; D002462:Cell Membrane / Q000378:metabolism; D015536:Down-Regulation; D027341:Excitatory Amino Acid Transporter 1 / Q000235:genetics / Q000378:metabolism*; D027342:Excitatory Amino Acid Transporter 2 / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression; D018698:Glutamic Acid / Q000378:metabolism; D006801:Humans; D009154:Mutation; D009865:Oocytes / Q000378:metabolism; D018408:Patch-Clamp Techniques; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D014982:Xenopus laevis
chemical_list: D027341:Excitatory Amino Acid Transporter 1; D027342:Excitatory Amino Acid Transporter 2; D018698:Glutamic Acid; C118135:OXSR1 protein, human; D017346:Protein Serine-Threonine Kinases; C486409:STK39 protein, human
keywords: Glutamate; Neuronal excitation; Oxidative stress-responsive kinase 1; SPS1-related proline/alanine-rich kinase; WNK