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 overexpressing the Thr287Asp mutant to enhance autonomous CaMKII activity had no effect on either EAAT1 or EAAT2 mediated glutamate uptake, overexpressing a dominant-negative version of CaMKII (Asp136Asn) diminished EAAT1 glutamate uptake. 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) 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.

CaMKII enhances voltage-gated sodium channel Nav1.6 activity and neuronal excitability

“…In these experiments, we compared the phosphorylation status of Nav1.6 in naive cells versus cells treated with CaMKII active inhibitors (KN93 overnight at 30°C and tatCN21 immediately before harvesting) or inactive compounds (KN92 and tatCN21Ala). Whereas we observed 21.5 6 1.6% autonomously active CaMKII in our Nav1.6-expressing HEK293 cells, which is similar to previously reported values (60), we also exposed these cells to the calcium ionophore ionomycin to promote CaMKII signaling (61) as well as exposing the immunopurified Nav1.6 to recombinant preautophosphorylated (activated) aCaMKII, two treatments that might be expected to maximize CaMKII phosphorylation of Nav1.6. Therefore, a phosphorylation site was considered to be CaMKII-dependent if phosphorylation of that site was observed by nanoflow LC-ESI/MS in both the ionomycin and activated aCaMKII conditions.…”

Activity dependent internalization of the glutamate transporter GLT-1 requires calcium entry through the NCX sodium/calcium exchanger

“…AMPK is also activated in ischemia (Li and McCullough, 2010) and it is known to stimulate Nedd4-2 (Bhalla et al, 2006). CamKII is less likely to fulfil an important role in ischemia as it only appears to effect the internalization of GLT-1b, a minor GLT-1 isoform, and not that of the major isoform, GLT-1a (Chawla et al, 2017;Underhill et al, 2015). Indeed, the CamKII inhibitor CN-93 did not impede the internalization of GLT-1 in astrocytes (data not shown).…”

Glutamate Transport: A New Bench to Bedside Mechanism for Treating Drug Abuse

“…Glutamate itself can rapidly increase GLAST activity by increasing surface expression ( Duan et al, 1999 ). Calcium/calmodulin (CAMKII)-dependent EAAT1 phosphorylation regulates constitutive transporter activity ( Chawla et al, 2017 ), and consensus sequences for Protein Kinase A (PKA), Protein Kinase C (PKC), and Phosphoinositide 3-Kinase (PI3K) suggest that phosphorylation may also rapidly modulate EAAT1 activity. PKA or PKC inhibitors reduce cell surface EAAT1 expression, while a PI3K inhibitor increases surface protein in primary cortical cultures ( Guillet et al, 2005 ).…”