Mechanisms Underlying CaMKII Regulation of Astrocytic Glutamate Transporters

Abstract

Excitotoxic calcium signaling, a hallmark of aberrant neuronal activity during cerebral ischemia and brain trauma, induces a loss of CaMKII activity that appears to sensitize neurons to glutamate-induced toxicity. Because CaMKII is present in astrocytes and other glia, we postulate that aberrant CaMKII signaling could compromise neuronal signaling and survival to excitotoxicity by disrupting astrocyte function. In support of this model, inhibition of CaMKII in cortical astrocytic cultures with a peptide (tat-CN21) as well as a small molecule (KN-93) inhibitor significantly reduces glutamate uptake compared to inactive controls, suggesting that glutamate transporters in astrocytes require CaMKII for their basal activity. The two primary excitatory amino acid transporters (EAAT) in astrocytes are EAAT 1 and EAAT2. Co-immunoprecipitation from rodent brain and immunocytochemical studies in cortical astrocytes reveal an association and co-localization respectively between EAAT2 and CaMKII. Although additional studies are required to determine if these interactions exist for EAAT1 and CaMKII in cells, in vitro studies using GST-fusion proteins and peptide tiling arrays indicate that purified δCaMKII binds to the N terminal and second intracellular loop of EAAT1, whereas, CaMKII preferentially binds to the N terminus and third intracellular loop of EAAT2. Using P32 incorporation as an index of CaMKII phosphorylation, phosphorylation of immunoprecipitated transporters as well as peptide tiling assays for EAAT2 reveal that astrocytic glutamate transporters are indeed CaMKII substrates. In total, our data support the hypothesis that astrocytic glutamate transporters are regulated by CaMKII binding and/or phosphorylation. Further studies are required to determine if a loss of CaMKII activity that is associated with pathological states, including epilepsy and ischemic stroke, compromises neuronal survival due to reduced glutamate transporter function previously associated with excitotoxicity.