Abstract
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.
Highlights
Astrocytes, the most numerous cells in the human brainstem and cortex [1], are essential for neuronal viability, in part, by maintaining extracellular homeostasis
Using a cell surface biotinylation assay, we demonstrate that TREK-2 protein levels were significantly increased in both cytoplasmic (2.36 fold ± 0.58 SEM) and membrane fractions (1.64 ± 0.25 SEM) obtained from astrocytes after 24 hours of hypoxia/hypoglycemic conditions (Fig 1)
These data confirm that the increased outward current in astrocytes exposed to ischemic conditions previously reported in Kucheryavykh et al [7] is due to a greater number of functional TREK-2 channels in the astrocytic membrane
Summary
Astrocytes, the most numerous cells in the human brainstem and cortex [1], are essential for neuronal viability, in part, by maintaining extracellular homeostasis. Posttranscriptional mechanisms could be involved in TREK-2 up-regulation These include: 1) regulation of translation by factors binding to the untranslated region (UTR) of mRNA [19], 2) liberation of TREK-2 mRNA from production bodies or p-bodies [20], 3) increased insertion of channel protein into the plasma membrane [21], and/or 4) decreased degradation of protein [22]. All of these processes would result in increased TREK-2 protein within the astrocyte. We provide in vivo evidence for up-regulation of astrocytic TREK-2 channels after middle cerebral artery occlusion (MCAO) using immunocytochemistry and immunoblotting
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