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KCa3.1/IK1 Channel Regulation by cGMP-Dependent Protein Kinase (PKG) via Reactive Oxygen Species and CaMKII in Microglia: An Immune Modulating Feedback System?

Ferreira R, Wong R, Schlichter LC - Front Immunol (2015)

Bottom Line: We previously found that KCa3.1 trafficking and gating require calmodulin (CaM) binding, and this is inhibited by cAMP kinase (PKA) acting at a single phosphorylation site.Similar results were seen in alternative-activated primary rat microglia; their KCa3.1 current required PKG, ROS, and CaMKII, and they had increased ROS production that required KCa3.1 activity.The increase in current apparently did not result from direct effects on the channel open probability (P o) or Ca(2+) dependence because, in inside-out patches from transfected HEK293 cells, single-channel activity was not affected by cGMP, PKG, H2O2 at normal or elevated intracellular Ca(2+).

View Article: PubMed Central - PubMed

Affiliation: Genetics and Development Division, Toronto Western Research Institute, University Health Network , Toronto, ON , Canada ; Department of Physiology, University of Toronto , Toronto, ON , Canada.

ABSTRACT
The intermediate conductance Ca(2+)-activated K(+) channel, KCa3.1 (IK1/SK4/KCNN4) is widely expressed in the innate and adaptive immune system. KCa3.1 contributes to proliferation of activated T lymphocytes, and in CNS-resident microglia, it contributes to Ca(2+) signaling, migration, and production of pro-inflammatory mediators (e.g., reactive oxygen species, ROS). KCa3.1 is under investigation as a therapeutic target for CNS disorders that involve microglial activation and T cells. However, KCa3.1 is post-translationally regulated, and this will determine when and how much it can contribute to cell functions. We previously found that KCa3.1 trafficking and gating require calmodulin (CaM) binding, and this is inhibited by cAMP kinase (PKA) acting at a single phosphorylation site. The same site is potentially phosphorylated by cGMP kinase (PKG), and in some cells, PKG can increase Ca(2+), CaM activation, and ROS. Here, we addressed KCa3.1 regulation through PKG-dependent pathways in primary rat microglia and the MLS-9 microglia cell line, using perforated-patch recordings to preserve intracellular signaling. Elevating cGMP increased both the KCa3.1 current and intracellular ROS production, and both were prevented by the selective PKG inhibitor, KT5823. The cGMP/PKG-evoked increase in KCa3.1 current in intact MLS-9 microglia was mediated by ROS, mimicked by applying hydrogen peroxide (H2O2), inhibited by a ROS scavenger (MGP), and prevented by a selective CaMKII inhibitor (mAIP). Similar results were seen in alternative-activated primary rat microglia; their KCa3.1 current required PKG, ROS, and CaMKII, and they had increased ROS production that required KCa3.1 activity. The increase in current apparently did not result from direct effects on the channel open probability (P o) or Ca(2+) dependence because, in inside-out patches from transfected HEK293 cells, single-channel activity was not affected by cGMP, PKG, H2O2 at normal or elevated intracellular Ca(2+). The regulation pathway we have identified in intact microglia and MLS-9 cells is expected to have broad implications because KCa3.1 plays important roles in numerous cells and tissues.

No MeSH data available.


Related in: MedlinePlus

Summary of results and proposed model of KCa3.1 regulation in microglia. Elevating intracellular cGMP activates PKG, which can then phosphorylate numerous downstream cellular targets, one of which triggers mitochondrial production of ROS (proposed to be via the “5-hydroxydecanoate-sensitive factor” that is likely the mitoKATP channel). Intracellular ROS can then contributes to KCa3.1 regulation through its role as a signaling intermediate; e.g., by evoking Ca2+ release from intracellular stores on the ER, leading to CaM-dependent activation of CaMKII, which then increases KCa3.1 activity (by an unknown mechanism). Activator used: 100 μM db-cGMP (membrane-permeant cGMP analog) to activate PKG. Inhibitors used: 1 μM KT5823 for PKG; 500 μM MPG as a general ROS scavenger (including O2–, H2O2, OH∙); 1 μM mAIP for CaMKII. Acronyms: CaMKII, Ca2+/CaM-dependent protein kinase II; cGMP, cyclic guanosine monophosphate; db-cGMP, dibutyryl-cyclic guanosine monophosphate; ER, endoplasmic reticulum; H2O2, hydrogen peroxide; mAIP, myristolated autocamtide-2 related inhibitory peptide; mitoKATP, mitochondrial ATP-sensitive potassium channel; MPG, N-(2-mercaptopropionyl)glycine; O2–, superoxide; OH∙, hydroxyl radical; PKG, cGMP-dependent protein kinase; ROS, reactive oxygen species.
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Figure 7: Summary of results and proposed model of KCa3.1 regulation in microglia. Elevating intracellular cGMP activates PKG, which can then phosphorylate numerous downstream cellular targets, one of which triggers mitochondrial production of ROS (proposed to be via the “5-hydroxydecanoate-sensitive factor” that is likely the mitoKATP channel). Intracellular ROS can then contributes to KCa3.1 regulation through its role as a signaling intermediate; e.g., by evoking Ca2+ release from intracellular stores on the ER, leading to CaM-dependent activation of CaMKII, which then increases KCa3.1 activity (by an unknown mechanism). Activator used: 100 μM db-cGMP (membrane-permeant cGMP analog) to activate PKG. Inhibitors used: 1 μM KT5823 for PKG; 500 μM MPG as a general ROS scavenger (including O2–, H2O2, OH∙); 1 μM mAIP for CaMKII. Acronyms: CaMKII, Ca2+/CaM-dependent protein kinase II; cGMP, cyclic guanosine monophosphate; db-cGMP, dibutyryl-cyclic guanosine monophosphate; ER, endoplasmic reticulum; H2O2, hydrogen peroxide; mAIP, myristolated autocamtide-2 related inhibitory peptide; mitoKATP, mitochondrial ATP-sensitive potassium channel; MPG, N-(2-mercaptopropionyl)glycine; O2–, superoxide; OH∙, hydroxyl radical; PKG, cGMP-dependent protein kinase; ROS, reactive oxygen species.

Mentions: Figure 7 summarizes our results and presents a model of KCa3.1 post-translational regulation based on our observations and the literature. Elevating cGMP activates PKG, which increases ROS production, evokes Ca2+ release from intracellular stores, which binds to CaM, and opens the KCa3.1 channel. CaM also activates CaMKII, which enhances the KCa3.1 current through an unknown mechanism. This is the first report of KCa3.1 regulation by cGMP/PKG and ROS. In comparing the present results with the literature, it is important to note several experimental considerations.


KCa3.1/IK1 Channel Regulation by cGMP-Dependent Protein Kinase (PKG) via Reactive Oxygen Species and CaMKII in Microglia: An Immune Modulating Feedback System?

Ferreira R, Wong R, Schlichter LC - Front Immunol (2015)

Summary of results and proposed model of KCa3.1 regulation in microglia. Elevating intracellular cGMP activates PKG, which can then phosphorylate numerous downstream cellular targets, one of which triggers mitochondrial production of ROS (proposed to be via the “5-hydroxydecanoate-sensitive factor” that is likely the mitoKATP channel). Intracellular ROS can then contributes to KCa3.1 regulation through its role as a signaling intermediate; e.g., by evoking Ca2+ release from intracellular stores on the ER, leading to CaM-dependent activation of CaMKII, which then increases KCa3.1 activity (by an unknown mechanism). Activator used: 100 μM db-cGMP (membrane-permeant cGMP analog) to activate PKG. Inhibitors used: 1 μM KT5823 for PKG; 500 μM MPG as a general ROS scavenger (including O2–, H2O2, OH∙); 1 μM mAIP for CaMKII. Acronyms: CaMKII, Ca2+/CaM-dependent protein kinase II; cGMP, cyclic guanosine monophosphate; db-cGMP, dibutyryl-cyclic guanosine monophosphate; ER, endoplasmic reticulum; H2O2, hydrogen peroxide; mAIP, myristolated autocamtide-2 related inhibitory peptide; mitoKATP, mitochondrial ATP-sensitive potassium channel; MPG, N-(2-mercaptopropionyl)glycine; O2–, superoxide; OH∙, hydroxyl radical; PKG, cGMP-dependent protein kinase; ROS, reactive oxygen species.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4389654&req=5

Figure 7: Summary of results and proposed model of KCa3.1 regulation in microglia. Elevating intracellular cGMP activates PKG, which can then phosphorylate numerous downstream cellular targets, one of which triggers mitochondrial production of ROS (proposed to be via the “5-hydroxydecanoate-sensitive factor” that is likely the mitoKATP channel). Intracellular ROS can then contributes to KCa3.1 regulation through its role as a signaling intermediate; e.g., by evoking Ca2+ release from intracellular stores on the ER, leading to CaM-dependent activation of CaMKII, which then increases KCa3.1 activity (by an unknown mechanism). Activator used: 100 μM db-cGMP (membrane-permeant cGMP analog) to activate PKG. Inhibitors used: 1 μM KT5823 for PKG; 500 μM MPG as a general ROS scavenger (including O2–, H2O2, OH∙); 1 μM mAIP for CaMKII. Acronyms: CaMKII, Ca2+/CaM-dependent protein kinase II; cGMP, cyclic guanosine monophosphate; db-cGMP, dibutyryl-cyclic guanosine monophosphate; ER, endoplasmic reticulum; H2O2, hydrogen peroxide; mAIP, myristolated autocamtide-2 related inhibitory peptide; mitoKATP, mitochondrial ATP-sensitive potassium channel; MPG, N-(2-mercaptopropionyl)glycine; O2–, superoxide; OH∙, hydroxyl radical; PKG, cGMP-dependent protein kinase; ROS, reactive oxygen species.
Mentions: Figure 7 summarizes our results and presents a model of KCa3.1 post-translational regulation based on our observations and the literature. Elevating cGMP activates PKG, which increases ROS production, evokes Ca2+ release from intracellular stores, which binds to CaM, and opens the KCa3.1 channel. CaM also activates CaMKII, which enhances the KCa3.1 current through an unknown mechanism. This is the first report of KCa3.1 regulation by cGMP/PKG and ROS. In comparing the present results with the literature, it is important to note several experimental considerations.

Bottom Line: We previously found that KCa3.1 trafficking and gating require calmodulin (CaM) binding, and this is inhibited by cAMP kinase (PKA) acting at a single phosphorylation site.Similar results were seen in alternative-activated primary rat microglia; their KCa3.1 current required PKG, ROS, and CaMKII, and they had increased ROS production that required KCa3.1 activity.The increase in current apparently did not result from direct effects on the channel open probability (P o) or Ca(2+) dependence because, in inside-out patches from transfected HEK293 cells, single-channel activity was not affected by cGMP, PKG, H2O2 at normal or elevated intracellular Ca(2+).

View Article: PubMed Central - PubMed

Affiliation: Genetics and Development Division, Toronto Western Research Institute, University Health Network , Toronto, ON , Canada ; Department of Physiology, University of Toronto , Toronto, ON , Canada.

ABSTRACT
The intermediate conductance Ca(2+)-activated K(+) channel, KCa3.1 (IK1/SK4/KCNN4) is widely expressed in the innate and adaptive immune system. KCa3.1 contributes to proliferation of activated T lymphocytes, and in CNS-resident microglia, it contributes to Ca(2+) signaling, migration, and production of pro-inflammatory mediators (e.g., reactive oxygen species, ROS). KCa3.1 is under investigation as a therapeutic target for CNS disorders that involve microglial activation and T cells. However, KCa3.1 is post-translationally regulated, and this will determine when and how much it can contribute to cell functions. We previously found that KCa3.1 trafficking and gating require calmodulin (CaM) binding, and this is inhibited by cAMP kinase (PKA) acting at a single phosphorylation site. The same site is potentially phosphorylated by cGMP kinase (PKG), and in some cells, PKG can increase Ca(2+), CaM activation, and ROS. Here, we addressed KCa3.1 regulation through PKG-dependent pathways in primary rat microglia and the MLS-9 microglia cell line, using perforated-patch recordings to preserve intracellular signaling. Elevating cGMP increased both the KCa3.1 current and intracellular ROS production, and both were prevented by the selective PKG inhibitor, KT5823. The cGMP/PKG-evoked increase in KCa3.1 current in intact MLS-9 microglia was mediated by ROS, mimicked by applying hydrogen peroxide (H2O2), inhibited by a ROS scavenger (MGP), and prevented by a selective CaMKII inhibitor (mAIP). Similar results were seen in alternative-activated primary rat microglia; their KCa3.1 current required PKG, ROS, and CaMKII, and they had increased ROS production that required KCa3.1 activity. The increase in current apparently did not result from direct effects on the channel open probability (P o) or Ca(2+) dependence because, in inside-out patches from transfected HEK293 cells, single-channel activity was not affected by cGMP, PKG, H2O2 at normal or elevated intracellular Ca(2+). The regulation pathway we have identified in intact microglia and MLS-9 cells is expected to have broad implications because KCa3.1 plays important roles in numerous cells and tissues.

No MeSH data available.


Related in: MedlinePlus