title: 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.
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.
authors.affiliation: Department of Biochemistry, Universidad Central del Caribe, Bayamón, Puerto Rico, United States of America.
authors.forename: Aixa F
authors.initials: AF
authors.lastname: Rivera-Pagán
authors.affiliation: Department of Biochemistry, Universidad Central del Caribe, Bayamón, Puerto Rico, United States of America.
authors.forename: David E
authors.initials: DE
authors.lastname: Rivera-Aponte
authors.affiliation: Department of Biochemistry, Universidad Central del Caribe, Bayamón, Puerto Rico, United States of America.
authors.forename: Katya V
authors.initials: KV
authors.lastname: Melnik-Martínez
authors.affiliation: Department of Physiology, Universidad Central del Caribe, Bayamón, Puerto Rico, United States of America.
authors.forename: Astrid
authors.initials: A
authors.lastname: Zayas-Santiago
authors.affiliation: Department of Biochemistry, Universidad Central del Caribe, Bayamón, Puerto Rico, United States of America.
authors.forename: Lilia Y
authors.initials: LY
authors.lastname: Kucheryavykh
authors.affiliation: Department of Biochemistry, Universidad Central del Caribe, Bayamón, Puerto Rico, United States of America.
authors.forename: Antonio H
authors.initials: AH
authors.lastname: Martins
authors.affiliation: Departments of Anatomy and Cell Biology, Universidad Central del Caribe, Bayamón, Puerto Rico, United States of America.
authors.forename: Luis A
authors.initials: LA
authors.lastname: Cubano
authors.affiliation: Department of Biochemistry, Universidad Central del Caribe, Bayamón, Puerto Rico, United States of America; Department of Physiology, Universidad Central del Caribe, Bayamón, Puerto Rico, United States of America.
authors.forename: Serguei N
authors.initials: SN
authors.lastname: Skatchkov
authors.affiliation: Department of Biochemistry, Universidad Central del Caribe, Bayamón, Puerto Rico, United States of America.
authors.forename: Misty J
authors.initials: MJ
authors.lastname: Eaton
mesh_terms: D000818:Animals; D001253:Astrocytes / Q000378:metabolism* / Q000473:pathology; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D005904:Glial Fibrillary Acidic Protein / Q000378:metabolism; D006801:Humans; D020244:Infarction, Middle Cerebral Artery / Q000378:metabolism / Q000473:pathology; D002546:Ischemic Attack, Transient / Q000378:metabolism* / Q000473:pathology; D024683:Potassium Channels, Tandem Pore Domain / Q000235:genetics / Q000378:metabolism*; D014176:Protein Biosynthesis; D017207:Rats, Sprague-Dawley; D015854:Up-Regulation
chemical_list: D005904:Glial Fibrillary Acidic Protein; C503444:Kcnk10 protein, rat; D024683:Potassium Channels, Tandem Pore Domain