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Constitutive regulation of the glutamate/aspartate transporter EAAT 1 by Calcium‐Calmodulin‐Dependent Protein Kinase II
Author(s) -
Chawla Aarti R.,
Johnson Derrick E.,
Zybura Agnes S.,
Leeds Benjamin P.,
Nelson Ross M.,
Hudmon Andy
Publication year - 2017
Publication title -
journal of neurochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.75
H-Index - 229
eISSN - 1471-4159
pISSN - 0022-3042
DOI - 10.1111/jnc.13913
Subject(s) - glutamate receptor , biology , glutamate aspartate transporter , excitatory postsynaptic potential , biochemistry , protein kinase a , phosphorylation , microbiology and biotechnology , metabotropic glutamate receptor , receptor
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 EAAT 1 and EAAT 2 ( GLAST and GLT ‐1, respectively, in rodents). While the inhibition of calcium/calmodulin‐dependent kinase (Ca MKII ), 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 Ca MKII regulation of EAAT 1 and EAAT 2. In transiently transfected HEK 293T cells, pharmacological inhibition of Ca MKII (using KN ‐93 or tat‐ CN 21) reduces [ 3 H]‐glutamate uptake in EAAT 1 without altering EAAT 2‐mediated glutamate uptake. While over‐expressing the Thr287Asp mutant to enhance autonomous Ca MKII activity had no effect on either EAAT 1 or EAAT 2‐mediated glutamate uptake, over‐expressing a dominant‐negative version of Ca MKII (Asp136Asn) diminished EAAT 1 glutamate uptake. SPOTS peptide arrays and recombinant glutathione S‐transferase‐fusion proteins of the intracellular N‐ and C‐termini of EAAT 1 identified two potential phosphorylation sites at residues Thr26 and Thr37 in the N‐terminus. Introducing an Ala (a non‐phospho mimetic) at Thr37 diminished EAAT 1‐mediated glutamate uptake, suggesting that the phosphorylation state of this residue is important for constitutive EAAT 1 function. Our study is the first to identify a glutamate transporter as a direct Ca MKII substrate and suggests that Ca MKII signaling is a critical driver of constitutive glutamate uptake by EAAT 1.