A-Kinase-Anchoring Protein (AKAP150) is expressed in Astrocytes and Upregulated in Response to Ischemia.

A-kinase-anchoring proteins, AKAPs, are scaffolding proteins that associate with kinases and phosphatases, and direct them to a specific submembrane site to coordinate signaling events. AKAP150, a rodent ortholog of human AKAP79, has been extensively studied in neurons, but very little is known about the localization and function of AKAP150 in astrocytes, the major cell type in brain. Thus, in this study, we assessed the localization of AKAP150 in astrocytes and elucidated its role during physiological and ischemic conditions. Herein, we demonstrate that AKAP150 is localized in astrocytes and is up-regulated during ischemia both in vitro and in vivo. Knock-down of AKAP150 by RNAi depolarizes the astrocytic membrane potential and substantially reduces by 80% the ability of astrocytes to take up extracellular potassium during ischemic conditions. Therefore, upregulation of AKAP150 during ischemia preserves potassium conductance and the associated hyperpolarized membrane potential of astrocytes; properties of astrocytes needed to maintain extracellular brain homeostasis. Taken together, these data suggest that AKAP150 may play a pivotal role in the neuroprotective mechanism of astrocytes during pathological conditions.

Up-regulation of TREK-2 potassium channels in cultured astrocytes requires de novo protein synthesis: relevance to localization of TREK-2 channels in astrocytes after transient cerebral ischemia.

Excitotoxicity due to glutamate receptor over-activation is one of the key mediators of neuronal death after an ischemic insult. Therefore, a major function of astrocytes is to maintain low extracellular levels of glutamate. The ability of astrocytic glutamate transporters to regulate the extracellular glutamate concentration depends upon the hyperpolarized membrane potential of astrocytes conferred by the presence of K+ channels in their membranes. We have previously shown that TREK-2 potassium channels in cultured astrocytes are up-regulated by ischemia and may support glutamate clearance by astrocytes during ischemia. Thus, herein we determine the mechanism leading to this up-regulation and assess the localization of TREK-2 channels in astrocytes after transient middle cerebral artery occlusion. By using a cell surface biotinylation assay we confirmed that functional TREK-2 protein is up-regulated in the astrocytic membrane after ischemic conditions. Using real time RT-PCR, we determined that the levels of TREK-2 mRNA were not increased in response to ischemic conditions. By using Western blot and a variety of protein synthesis inhibitors, we demonstrated that the increase of TREK-2 protein expression requires De novo protein synthesis, while protein degradation pathways do not contribute to TREK-2 up-regulation after ischemic conditions. Immunohistochemical studies revealed TREK-2 localization in astrocytes together with increased expression of the selective glial marker, glial fibrillary acidic protein, in brain 24 hours after transient middle cerebral occlusion. Our data indicate that functional TREK-2 channels are up-regulated in the astrocytic membrane during ischemia through a mechanism requiring De novo protein synthesis. This study provides important information about the mechanisms underlying TREK-2 regulation, which has profound implications in neurological diseases such as ischemia where astrocytes play an important role.