title: SPAK and OSR1 Sensitivity of Excitatory Amino Acid Transporter EAAT3.
abstract: OBJECTIVE
  Kinases involved in the regulation of epithelial transport include SPAK (SPS1-related proline/alanine-rich kinase) and OSR1 (oxidative stress-responsive kinase 1). SPAK and OSR1 are both regulated by WNK (with-no-K(Lys)) kinases. The present study explored whether SPAK and/or OSR1 influence the excitatory amino acid transporter EAAT3, which accomplishes glutamate and aspartate transport in kidney, intestine and brain.
  
  
  METHODS
  cRNA encoding EAAT3 was injected into Xenopus laevis oocytes with or without additional injection of cRNA encoding wild-type SPAK, constitutively active (T233E)SPAK, WNK insensitive (T233A)SPAK, catalytically inactive (D212A)SPAK, wild-type OSR1, constitutively active (T185E)OSR1, WNK insensitive (T185A)OSR1 and catalytically inactive (D164A)OSR1. Glutamate-induced current was taken as measure of electrogenic glutamate transport and was quantified utilizing dual electrode voltage clamp. Furthermore, Ussing chamber was employed to determine glutamate transport in the intestine from gene-targeted mice carrying WNK insensitive SPAK (spak(tg/tg)) and from corresponding wild-type mice (spak(+/+)).
  
  
  RESULTS
  EAAT3 activity was significantly decreased by wild-type SPAK and (T233E)SPAK, but not by (T233A)SPAK and (D212A)SPAK. SPAK decreased maximal transport rate without affecting significantly affinity of the carrier. Similarly, EAAT3 activity was significantly downregulated by wild-type OSR1 and (T185E)OSR1, but not by (T185A)OSR1 and (D164A)OSR1. Again OSR1 decreased maximal transport rate without affecting significantly affinity of the carrier. Intestinal electrogenic glutamate transport was significantly lower in spak(+/+) than in spak(tg/tg) mice.
  
  
  CONCLUSIONS
  Both, SPAK and OSR1 are negative regulators of EAAT3 activity.
authors.affiliation: Department of Physiology I, University of Tübingen, Tubingen, Germany.
authors.forename: José
authors.initials: J
authors.lastname: Borrás
authors.forename: Madhuri S
authors.initials: MS
authors.lastname: Salker
authors.forename: Bernat
authors.initials: B
authors.lastname: Elvira
authors.forename: Jamshed
authors.initials: J
authors.lastname: Warsi
authors.forename: Myriam
authors.initials: M
authors.lastname: Fezai
authors.forename: Zohreh
authors.initials: Z
authors.lastname: Hoseinzadeh
authors.forename: Florian
authors.initials: F
authors.lastname: Lang
mesh_terms: D000818:Animals; D001224:Aspartic Acid / Q000378:metabolism; D050580:Excitatory Amino Acid Transporter 3 / Q000235:genetics / Q000378:metabolism*; D018698:Glutamic Acid / Q000378:metabolism; D006801:Humans; D051379:Mice; D009865:Oocytes / Q000378:metabolism; D018408:Patch-Clamp Techniques; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D018075:RNA, Complementary / Q000096:biosynthesis / Q000235:genetics; D014867:Water / Q000378:metabolism; D014982:Xenopus laevis
chemical_list: D050580:Excitatory Amino Acid Transporter 3; D018075:RNA, Complementary; C493400:SLC1A1 protein, human; D014867:Water; D001224:Aspartic Acid; D018698:Glutamic Acid; C118135:OXSR1 protein, human; D017346:Protein Serine-Threonine Kinases; C486409:STK39 protein, human