Down-regulation of Na+-coupled glutamate transporter EAAT3 and EAAT4 by AMP-activated protein kinase.
The glutamate transporters EAAT3 and EAAT4 are expressed in neurons. They contribute to the cellular uptake of glutamate and aspartate and thus to the clearance of the excitatory transmitters from the extracellular space. During ischemia, extracellular accumulation of glutamate may trigger excitotoxicity. Energy depletion leads to activation of the AMP-activated protein kinase (AMPK), a kinase enhancing energy production and limiting energy expenditure. The present study thus explored the possibility that AMPK regulates EAAT3 and/or EAAT4. To this end, EAAT3 or EAAT4 were expressed in Xenopus oocytes with or without AMPK and electrogenic glutamate transport determined by dual electrode voltage clamp. In EAAT3- and in EAAT4-expressing oocytes glutamate generated a current (I(g)), which was half maximal (K(M)) at 74 microM (EAAT3) or at 4 microM (EAAT4) glutamate. Co-expression of constitutively active (gammaR70Q)AMPK or of wild type AMPK did not affect K(M) but significantly decreased the maximal I(g) in both EAAT3- (by 34%) and EAAT4- (by 49%) expressing oocytes. Co-expression of the inactive mutant (alphaK45R)AMPK [alpha1(K45R)beta1gamma1] did not appreciably affect I(g). According to confocal microscopy and chemiluminescence co-expression of (gammaR70Q)AMPK or of wild type AMPK reduced the membrane abundance of EAAT3 and EAAT4. The observations show that AMPK down-regulates Na(+)-coupled glutamate transport.
Sopjani
Mentor
M
Department of Physiology, University of Tübingen, Tübingen, Germany.
Alesutan
Ioana
I
Dërmaku-Sopjani
Miribane
M
Fraser
Scott
S
Kemp
Bruce E
BE
Föller
Michael
M
Lang
Florian
F
D055372:AMP-Activated Protein Kinases / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D001120:Arginine / Q000235:genetics; D004305:Dose-Response Relationship, Drug; D015536:Down-Regulation / Q000187:drug effects / Q000502:physiology*; D056827:Endosomal Sorting Complexes Required for Transport / Q000494:pharmacology; D050580:Excitatory Amino Acid Transporter 3 / Q000235:genetics / Q000378:metabolism*; D050581:Excitatory Amino Acid Transporter 4 / Q000235:genetics / Q000378:metabolism*; D018698:Glutamic Acid / Q000494:pharmacology; D005973:Glutamine / Q000235:genetics; D006801:Humans; D008564:Membrane Potentials / Q000187:drug effects / Q000235:genetics; D018613:Microscopy, Confocal / Q000379:methods; D009154:Mutation / Q000235:genetics; D000075702:Nedd4 Ubiquitin Protein Ligases; D009589:Nitrogen Oxides / Q000494:pharmacology; D009865:Oocytes; D018408:Patch-Clamp Techniques / Q000379:methods; D014161:Transduction, Genetic; D014443:Tyrosine / Q000235:genetics; D044767:Ubiquitin-Protein Ligases / Q000494:pharmacology; D014981:Xenopus
D056827:Endosomal Sorting Complexes Required for Transport; D050580:Excitatory Amino Acid Transporter 3; D050581:Excitatory Amino Acid Transporter 4; D009589:Nitrogen Oxides; C493400:SLC1A1 protein, human; C493391:SLC1A6 protein, human; D005973:Glutamine; D018698:Glutamic Acid; D014443:Tyrosine; D001120:Arginine; D000075702:Nedd4 Ubiquitin Protein Ligases; D044767:Ubiquitin-Protein Ligases; D055372:AMP-Activated Protein Kinases