Down-Regulation of Excitatory Amino Acid Transporters EAAT1 and EAAT2 by the Kinases SPAK and OSR1
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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 T233ESPAK, WNK insensitive T233ASPAK, catalytically inactive D212ASPAK, constitutively active T185EOSR1, WNK insensitive T185AOSR1 or catalytically inactive D164AOSR1. 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 T233ESPAK and T185EOSR1, but not by T233ASPAK, D212ASPAK, T185AOSR1, or D164AOSR1. 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.
KeywordsNeuronal excitation Glutamate Oxidative stress-responsive kinase 1 SPS1-related proline/alanine-rich kinase WNK
The authors acknowledge the meticulous preparation of the manuscript by Tanja Loch and technical support by Elfriede Faber. This study was supported by the Deutsche Forschungsgemeinschaft, GRK 1302, SFB 773 B4/A1, La 315/13-3.
Conception and design of research: FL. Performed experiments AA, BE, IA. Analyzed data: AA, JW, BE, IA, ZH. Interpreted results of experiments: AA, JW, BE, ZH, FL. Prepared figures: AA, JW, BE, ZH, Drafted manuscript: FL. Edited and wrote manuscript: FL. Approved final version: AA, JW, BE, ZH, FL.
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Conflict of Interests
The authors of this manuscript state that they do not have any conflict of interests and nothing to disclose.
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