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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.

Rivera-Pagán AF, Rivera-Aponte DE, Melnik-Martínez KV, Zayas-Santiago A, Kucheryavykh LY, Martins AH, Cubano LA, Skatchkov SN, Eaton MJ - PLoS ONE (2015)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Universidad Central del Caribe, Bayamón, Puerto Rico, United States of America.

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.

No MeSH data available.


Related in: MedlinePlus

TREK-2 protein degradation pathway(s) are not altered after ischemia.Cortical astrocyte in cultures exposed to (A) control or (B) hypoxia/hypoglycemic conditions for 24 hours were treated with either 10 μM chloroquine, 50 μM calpeptin or 10 μM MG132 for 8 hours. Astrocytes were initially exposed to hypoxia/hypoglycemic conditions for 16 hours. The cells were then treated with the degradative pathway inhibitors or without treatment and returned to hypoxia/hypoglycemic conditions for an additional 8 hours. TREK-2 expression in astrocytes was determined by Western blot. The results of 3 separate experiments using different astrocyte cultures are shown. Data are expressed relative to control.
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pone.0125195.g003: TREK-2 protein degradation pathway(s) are not altered after ischemia.Cortical astrocyte in cultures exposed to (A) control or (B) hypoxia/hypoglycemic conditions for 24 hours were treated with either 10 μM chloroquine, 50 μM calpeptin or 10 μM MG132 for 8 hours. Astrocytes were initially exposed to hypoxia/hypoglycemic conditions for 16 hours. The cells were then treated with the degradative pathway inhibitors or without treatment and returned to hypoxia/hypoglycemic conditions for an additional 8 hours. TREK-2 expression in astrocytes was determined by Western blot. The results of 3 separate experiments using different astrocyte cultures are shown. Data are expressed relative to control.

Mentions: Regulation of protein degradation plays an active role in determining levels of proteins in a cell [30–32]. There are 3 major degradative pathways in mammalian cells, the Ubiquitin proteasome pathway, the Lysosomal pathway and the Calpain pathway. Thus, we next determined the normal degradation pathway(s) for TREK-2 within astrocytes during control or hypoxia/hypoglycemic conditions. Astrocytes exposed to control or hypoxia/hypoglycemic conditions for 24 hours and were incubated for the last 8 hours with either a proteasome inhibitor (10μM MG132), a lysosomal inhibitor (10μM chloroquine) or a calpain inhibitor (50μM calpeptin) after which the cells were harvested for Western blot. Control cells were run in parallel and incubated for 8 hours after addition of equal volume of the BBSS vehicle used for inhibitors. Inhibition of the pathway/pathways involved in TREK-2 degradation will result in increased TREK-2 protein within the cell. Fig 3A shows that the ubiquitin, lysosomal and calpain degradation pathways all seem to be involved in the regulation of TREK-2 protein degradation during control conditions, though these data could not be confirmed statistically. The relative chemiluminescence as compared to control was 2.09 ± 0.40, 1.92 ± 0.78 and 1.62 ± 0.62 SEM for chloroquine, calpeptin and MG132, respectively. When we examined if protein degradation plays a role during hypoxic/hypoglycemic conditions we found that the contribution of the different protein degradation pathways was not altered during ischemia, i.e., all three pathways were still being utilized for TREK-2 protein degradation (Fig 3B). The relative chemiluminescence as compared to control was 2.04 ± 0.57, 2.31 ± 0.71 and 2.26 ± 0.87 SEM for chloroquine, calpeptin and MG132, respectively.


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.

Rivera-Pagán AF, Rivera-Aponte DE, Melnik-Martínez KV, Zayas-Santiago A, Kucheryavykh LY, Martins AH, Cubano LA, Skatchkov SN, Eaton MJ - PLoS ONE (2015)

TREK-2 protein degradation pathway(s) are not altered after ischemia.Cortical astrocyte in cultures exposed to (A) control or (B) hypoxia/hypoglycemic conditions for 24 hours were treated with either 10 μM chloroquine, 50 μM calpeptin or 10 μM MG132 for 8 hours. Astrocytes were initially exposed to hypoxia/hypoglycemic conditions for 16 hours. The cells were then treated with the degradative pathway inhibitors or without treatment and returned to hypoxia/hypoglycemic conditions for an additional 8 hours. TREK-2 expression in astrocytes was determined by Western blot. The results of 3 separate experiments using different astrocyte cultures are shown. Data are expressed relative to control.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4401746&req=5

pone.0125195.g003: TREK-2 protein degradation pathway(s) are not altered after ischemia.Cortical astrocyte in cultures exposed to (A) control or (B) hypoxia/hypoglycemic conditions for 24 hours were treated with either 10 μM chloroquine, 50 μM calpeptin or 10 μM MG132 for 8 hours. Astrocytes were initially exposed to hypoxia/hypoglycemic conditions for 16 hours. The cells were then treated with the degradative pathway inhibitors or without treatment and returned to hypoxia/hypoglycemic conditions for an additional 8 hours. TREK-2 expression in astrocytes was determined by Western blot. The results of 3 separate experiments using different astrocyte cultures are shown. Data are expressed relative to control.
Mentions: Regulation of protein degradation plays an active role in determining levels of proteins in a cell [30–32]. There are 3 major degradative pathways in mammalian cells, the Ubiquitin proteasome pathway, the Lysosomal pathway and the Calpain pathway. Thus, we next determined the normal degradation pathway(s) for TREK-2 within astrocytes during control or hypoxia/hypoglycemic conditions. Astrocytes exposed to control or hypoxia/hypoglycemic conditions for 24 hours and were incubated for the last 8 hours with either a proteasome inhibitor (10μM MG132), a lysosomal inhibitor (10μM chloroquine) or a calpain inhibitor (50μM calpeptin) after which the cells were harvested for Western blot. Control cells were run in parallel and incubated for 8 hours after addition of equal volume of the BBSS vehicle used for inhibitors. Inhibition of the pathway/pathways involved in TREK-2 degradation will result in increased TREK-2 protein within the cell. Fig 3A shows that the ubiquitin, lysosomal and calpain degradation pathways all seem to be involved in the regulation of TREK-2 protein degradation during control conditions, though these data could not be confirmed statistically. The relative chemiluminescence as compared to control was 2.09 ± 0.40, 1.92 ± 0.78 and 1.62 ± 0.62 SEM for chloroquine, calpeptin and MG132, respectively. When we examined if protein degradation plays a role during hypoxic/hypoglycemic conditions we found that the contribution of the different protein degradation pathways was not altered during ischemia, i.e., all three pathways were still being utilized for TREK-2 protein degradation (Fig 3B). The relative chemiluminescence as compared to control was 2.04 ± 0.57, 2.31 ± 0.71 and 2.26 ± 0.87 SEM for chloroquine, calpeptin and MG132, respectively.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Universidad Central del Caribe, Bayamón, Puerto Rico, United States of America.

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.

No MeSH data available.


Related in: MedlinePlus