Indiana University-Purdue University Indianapolis (IUPUI) / Glutamate clearance by astrocytes is an essential part of physiological 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 (EAATs) in human astrocytes are EAAT1
and EAAT2 (GLAST and GLT-1 respectively in rodents). While the inhibition of a
ubiquitously-expressed serine/threonine protein kinase, the calcium/calmodulindependent
kinase (CaMKII) results in diminished glutamate uptake in cultured primary
rodent astrocytes, the molecular mechanism underlying this regulation is unknown. In
order to delineate this mechanism, we use a heterologous expression model to explore
CaMKII regulation of EAAT1 and EAAT2. In transiently transfected HEK293T cells,
pharmacological inhibition of CaMKII and overexpression of a dominant-negative
version of CaMKII (Asp136Asn) reduces [3H]-glutamate uptake by EAAT1, without
altering EAAT2 mediated glutamate uptake. Surprisingly, overexpression of a
constitutively active autophosphorylation mutant (Thr287Asp) to increase autonomous
CaMKII activity and a mutant incapable of autophosphorylation (Thr287Val) had no
effect on either EAAT1 or EAAT2 mediated glutamate uptake. Pulldown of FLAGtagged
glutamate transporters suggests CaMKII does not interact with EAAT1 or
EAAT2. SPOTS peptide arrays and recombinant GST-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) but not an Asp (phosphomimetic) at Thr37 diminished EAAT1-mediated glutamate uptake,
suggesting that the phosphorylation state of this residue is important for constitutive
EAAT1 function. In sum, this is the first report of a glutamate transporter being identified
as a direct CaMKII substrate. These findings indicate that CaMKII signaling is a critical
driver of homeostatic glutamate uptake by EAAT1. Aberrations in basal CaMKII activity
disrupt glutamate uptake, which can perpetuate glutamate-mediated excitotoxicity and
result in cellular death.
| Identifer | oai:union.ndltd.org:IUPUI/oai:scholarworks.iupui.edu:1805/10605 |
| Date | 19 May 2016 |
| Creators | Chawla, Aarti R. |
| Contributors | Hudmon, Andy, Cummins, Theodore, Oxford, Gerry S., Chen, Jinhui, Hoang, Quyen |
| Source Sets | Indiana University-Purdue University Indianapolis |
| Language | en_US |
| Detected Language | English |
| Type | Dissertation |
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