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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 expression is increased in the astrocytic membrane and cytoplasm after ischemia.Cortical astrocytes in culture were exposed to hypoxic/hypoglycemic conditions for 24 hours and then processed using a cell surface biotinylation assay. The graph displays the quantification of the relative chemiluminescence intensity ± standard error of the mean (SEM) of TREK-2 protein in membrane and cytoplasmic fractions obtained from control astrocytes and astrocytes subjected to ischemia (representative Western blots shown below the graph). TREK-2 was detected as a band of around 60kDa which is consistent with the predicted molecular weight of 59.6kDa. The results of 4 separate experiments using different astrocyte cultures are shown. The asterisks indicate a significant difference from control (t-test; p<0.05). Data are expressed relative to control.
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pone.0125195.g001: TREK-2 expression is increased in the astrocytic membrane and cytoplasm after ischemia.Cortical astrocytes in culture were exposed to hypoxic/hypoglycemic conditions for 24 hours and then processed using a cell surface biotinylation assay. The graph displays the quantification of the relative chemiluminescence intensity ± standard error of the mean (SEM) of TREK-2 protein in membrane and cytoplasmic fractions obtained from control astrocytes and astrocytes subjected to ischemia (representative Western blots shown below the graph). TREK-2 was detected as a band of around 60kDa which is consistent with the predicted molecular weight of 59.6kDa. The results of 4 separate experiments using different astrocyte cultures are shown. The asterisks indicate a significant difference from control (t-test; p<0.05). Data are expressed relative to control.

Mentions: TREK-2 protein and mRNA expression have been reported in cortical and hippocampal astrocytes [5,6,12] and TREK-2 protein levels together with TREK-like electrical currents are up-regulated after hypoxic/hypoglycemic conditions [7]. Therefore, we questioned if TREK-2 channel proteins are located in cytoplasmic vesicles or within the membrane of the astrocytes? This is an important issue since tandem-pore domain potassium channels maintain glial membrane potential and function [24,25] and specifically TREK-2 in astrocytes [7]. Using a cell surface biotinylation assay, we now 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. Although the mechanism involved in TREK-2 channel up-regulation is still unknown.


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 expression is increased in the astrocytic membrane and cytoplasm after ischemia.Cortical astrocytes in culture were exposed to hypoxic/hypoglycemic conditions for 24 hours and then processed using a cell surface biotinylation assay. The graph displays the quantification of the relative chemiluminescence intensity ± standard error of the mean (SEM) of TREK-2 protein in membrane and cytoplasmic fractions obtained from control astrocytes and astrocytes subjected to ischemia (representative Western blots shown below the graph). TREK-2 was detected as a band of around 60kDa which is consistent with the predicted molecular weight of 59.6kDa. The results of 4 separate experiments using different astrocyte cultures are shown. The asterisks indicate a significant difference from control (t-test; p<0.05). Data are expressed relative to control.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0125195.g001: TREK-2 expression is increased in the astrocytic membrane and cytoplasm after ischemia.Cortical astrocytes in culture were exposed to hypoxic/hypoglycemic conditions for 24 hours and then processed using a cell surface biotinylation assay. The graph displays the quantification of the relative chemiluminescence intensity ± standard error of the mean (SEM) of TREK-2 protein in membrane and cytoplasmic fractions obtained from control astrocytes and astrocytes subjected to ischemia (representative Western blots shown below the graph). TREK-2 was detected as a band of around 60kDa which is consistent with the predicted molecular weight of 59.6kDa. The results of 4 separate experiments using different astrocyte cultures are shown. The asterisks indicate a significant difference from control (t-test; p<0.05). Data are expressed relative to control.
Mentions: TREK-2 protein and mRNA expression have been reported in cortical and hippocampal astrocytes [5,6,12] and TREK-2 protein levels together with TREK-like electrical currents are up-regulated after hypoxic/hypoglycemic conditions [7]. Therefore, we questioned if TREK-2 channel proteins are located in cytoplasmic vesicles or within the membrane of the astrocytes? This is an important issue since tandem-pore domain potassium channels maintain glial membrane potential and function [24,25] and specifically TREK-2 in astrocytes [7]. Using a cell surface biotinylation assay, we now 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. Although the mechanism involved in TREK-2 channel up-regulation is still unknown.

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