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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 is up-regulated in vivo in astrocytes after tMCAO.(A) Expression changes of TREK-2 channel protein in brain after tMCAO. The results of 4 separate tMCAO experiments are shown. The asterisk indicates a significant difference from contralateral side (control) ± SEM (t-test; p<0.05). (B) TTC staining delineates a clearly detectable lesion at the infarct core (left side) at 24 hours after 60 minutes of tMCAO. (C) Immunostaining for TREK-2 (green labeling) and GFAP (red labeling) in hippocampus after tMCAO. Representative images show a qualitative increase of TREK-2 levels in hippocampus (C) on the ipsilateral (lesion) side of the brain. White arrows point to astrocytic processes and endfeet. Insets show higher magnification of the merged image to highlight colocalization between GFAP and TREK-2 channels in astrocytes.
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pone.0125195.g004: TREK-2 protein is up-regulated in vivo in astrocytes after tMCAO.(A) Expression changes of TREK-2 channel protein in brain after tMCAO. The results of 4 separate tMCAO experiments are shown. The asterisk indicates a significant difference from contralateral side (control) ± SEM (t-test; p<0.05). (B) TTC staining delineates a clearly detectable lesion at the infarct core (left side) at 24 hours after 60 minutes of tMCAO. (C) Immunostaining for TREK-2 (green labeling) and GFAP (red labeling) in hippocampus after tMCAO. Representative images show a qualitative increase of TREK-2 levels in hippocampus (C) on the ipsilateral (lesion) side of the brain. White arrows point to astrocytic processes and endfeet. Insets show higher magnification of the merged image to highlight colocalization between GFAP and TREK-2 channels in astrocytes.

Mentions: Using Western blot, we assessed the expression changes of TREK-2 channels in rat brain after transient middle cerebral artery occlusion (tMCAO). Fig 4A shows that TREK-2 protein was up-regulated (1.46 fold ± 0.052 SEM) on the ipsilateral side of the brain receiving the ischemic insult (left side) as compared with the contralateral side (right side). These data correlate with what has been previously reported by Li et al. [17], where they showed that TREK-2 expression was significantly increased in cortex and hippocampus 24 hours after tMCAO. Nevertheless, the authors concluded that TREK-2 up-regulation occurred in cortical and hippocampal neurons, but did not assess the effect of ischemia on astrocytes in the brain. Our next series of experiments directly addressed this question.


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 is up-regulated in vivo in astrocytes after tMCAO.(A) Expression changes of TREK-2 channel protein in brain after tMCAO. The results of 4 separate tMCAO experiments are shown. The asterisk indicates a significant difference from contralateral side (control) ± SEM (t-test; p<0.05). (B) TTC staining delineates a clearly detectable lesion at the infarct core (left side) at 24 hours after 60 minutes of tMCAO. (C) Immunostaining for TREK-2 (green labeling) and GFAP (red labeling) in hippocampus after tMCAO. Representative images show a qualitative increase of TREK-2 levels in hippocampus (C) on the ipsilateral (lesion) side of the brain. White arrows point to astrocytic processes and endfeet. Insets show higher magnification of the merged image to highlight colocalization between GFAP and TREK-2 channels in astrocytes.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4401746&req=5

pone.0125195.g004: TREK-2 protein is up-regulated in vivo in astrocytes after tMCAO.(A) Expression changes of TREK-2 channel protein in brain after tMCAO. The results of 4 separate tMCAO experiments are shown. The asterisk indicates a significant difference from contralateral side (control) ± SEM (t-test; p<0.05). (B) TTC staining delineates a clearly detectable lesion at the infarct core (left side) at 24 hours after 60 minutes of tMCAO. (C) Immunostaining for TREK-2 (green labeling) and GFAP (red labeling) in hippocampus after tMCAO. Representative images show a qualitative increase of TREK-2 levels in hippocampus (C) on the ipsilateral (lesion) side of the brain. White arrows point to astrocytic processes and endfeet. Insets show higher magnification of the merged image to highlight colocalization between GFAP and TREK-2 channels in astrocytes.
Mentions: Using Western blot, we assessed the expression changes of TREK-2 channels in rat brain after transient middle cerebral artery occlusion (tMCAO). Fig 4A shows that TREK-2 protein was up-regulated (1.46 fold ± 0.052 SEM) on the ipsilateral side of the brain receiving the ischemic insult (left side) as compared with the contralateral side (right side). These data correlate with what has been previously reported by Li et al. [17], where they showed that TREK-2 expression was significantly increased in cortex and hippocampus 24 hours after tMCAO. Nevertheless, the authors concluded that TREK-2 up-regulation occurred in cortical and hippocampal neurons, but did not assess the effect of ischemia on astrocytes in the brain. Our next series of experiments directly addressed this question.

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