title: Constitutive regulation of the glutamate/aspartate transporter EAAT1 by Calcium-Calmodulin-Dependent Protein Kinase II.
abstract: Glutamate clearance by astrocytes is an essential part of normal excitatory neurotransmission. Failure to adapt or maintain low levels of glutamate in the central nervous system is associated with multiple acute and chronic neurodegenerative diseases. The primary excitatory amino acid transporters in human astrocytes are EAAT1 and EAAT2 (GLAST and GLT-1, respectively, in rodents). While the inhibition of calcium/calmodulin-dependent kinase (CaMKII), a ubiquitously expressed serine/threonine protein kinase, results in diminished glutamate uptake in cultured primary rodent astrocytes (Ashpole et al. 2013), the molecular mechanism underlying this regulation is unknown. Here, we use a heterologous expression model to explore CaMKII regulation of EAAT1 and EAAT2. In transiently transfected HEK293T cells, pharmacological inhibition of CaMKII (using KN-93 or tat-CN21) reduces [3H]-glutamate uptake in EAAT1 without altering EAAT2-mediated glutamate uptake. While over-expressing the Thr287Asp mutant to enhance autonomous CaMKII activity had no effect on either EAAT1 or EAAT2-mediated glutamate uptake, over-expressing a dominant-negative version of CaMKII (Asp136Asn) diminished EAAT1 glutamate uptake. SPOTS peptide arrays and recombinant glutathione S-transferase-fusion proteins of the intracellular N- and C-termini of EAAT1 identified two potential phosphorylation sites at residues Thr26 and Thr37 in the N-terminus. Introducing an Ala (a non-phospho mimetic) at Thr37 diminished EAAT1-mediated glutamate uptake, suggesting that the phosphorylation state of this residue is important for constitutive EAAT1 function. Our study is the first to identify a glutamate transporter as a direct CaMKII substrate and suggests that CaMKII signaling is a critical driver of constitutive glutamate uptake by EAAT1.
authors.affiliation: Program in Medical Neuroscience, Indiana University School of Medicine, Indianapolis, Indiana, USA.|Paul and Carole Stark Neurosciences Research Institute, Indiana University School of Medicine, Indianapolis, Indiana, USA.
authors.forename: Aarti R
authors.initials: AR
authors.lastname: Chawla
authors.affiliation: Biochemistry Department, Indiana University School of Medicine, Indianapolis, Indiana, USA.
authors.forename: Derrick E
authors.initials: DE
authors.lastname: Johnson
authors.affiliation: Program in Medical Neuroscience, Indiana University School of Medicine, Indianapolis, Indiana, USA.|Paul and Carole Stark Neurosciences Research Institute, Indiana University School of Medicine, Indianapolis, Indiana, USA.
authors.forename: Agnes S
authors.initials: AS
authors.lastname: Zybura
authors.affiliation: Indiana University-Purdue University Indianapolis, Indianapolis, Indiana, USA.
authors.forename: Benjamin P
authors.initials: BP
authors.lastname: Leeds
authors.affiliation: Indiana University-Purdue University Indianapolis, Indianapolis, Indiana, USA.
authors.forename: Ross M
authors.initials: RM
authors.lastname: Nelson
authors.affiliation: Program in Medical Neuroscience, Indiana University School of Medicine, Indianapolis, Indiana, USA.|Paul and Carole Stark Neurosciences Research Institute, Indiana University School of Medicine, Indianapolis, Indiana, USA.|Biochemistry Department, Indiana University School of Medicine, Indianapolis, Indiana, USA.
authors.forename: Andy
authors.initials: A
authors.lastname: Hudmon
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001224:Aspartic Acid / Q000378:metabolism*; D001596:Benzylamines / Q000494:pharmacology; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2 / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D002478:Cells, Cultured; D027341:Excitatory Amino Acid Transporter 1 / Q000235:genetics / Q000378:metabolism*; D005260:Female; D018698:Glutamic Acid / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D008297:Male; D051381:Rats; D017207:Rats, Sprague-Dawley; D013449:Sulfonamides / Q000494:pharmacology
chemical_list: D001596:Benzylamines; D027341:Excitatory Amino Acid Transporter 1; C425780:KN 92; C493394:SLC1A3 protein, human; D013449:Sulfonamides; D001224:Aspartic Acid; D018698:Glutamic Acid; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2
keywords: 
  GLAST
  ; GLT-1; astrocytes; glutamate uptake