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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

PKG increases production of reactive oxygen species (ROS), which activates KCa3.1 current through a Ca2+ and CaMKII-mediated pathway. (A) Summarized data showing ROS production by unstimulated MLS-9 microglial cells (control), and after treatment (20 min; 37°C) with db-cGMP (100 μM), with or without the PKG inhibitor, KT5823 (1 μM). Values are expressed as mean ± SEM (n = 5), and compared using a one-way ANOVA with Tukey’s post hoc test. ***p < 0.001 treatment versus control; †††p < 0.001 with and without PKG inhibitor. (B) Acute application of db-cGMP increases intracellular Ca2+ in MLS-9 cells but does not activate KCa3.1 current. Upper panel: Representative Fura-2 recording, in which 100 μM db-cGMP was bath applied during the period marked by the horizontal bar. The inset shows calibrated free intracellular Ca2+ concentration as mean ± SEM, n = 19 cells (***p < 0.001, Student’s t-test). Lower panel: Representative time course of current in a perforated-patch recording (same solutions and voltage protocols as Figure 1) in which db-cGMP (100 μM) was bath applied as indicated by the horizontal bar. (C) KCa3.1 current potentiation by db-cGMP is prevented by the ROS scavenger, MPG, and the CaMKII inhibitor, mAIP. Each set of three traces shows representative currents before and after adding 300 μM riluzole, with or without 1 μM TRAM-34. Upper panel: Representative recording from an MLS-9 cell pre-treated with 100 μM db-cGMP and the ROS scavenger, MPG (500 μM), for 20 min at room temperature. Lower panel: Cells were pre-treated with 100 μM db-cGMP and the CaM kinase II inhibitor, mAIP (1 μM) for 20 min at room temperature. (D) Summarized data from a population study with treatments as in panel C. The TRAM-34-sensitive KCa3.1 current is expressed as mean ± SEM for the number of cells indicated on each bar, and was compared using a one-way ANOVA with Tukey’s post hoc test; ***p < 0.001.
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Figure 3: PKG increases production of reactive oxygen species (ROS), which activates KCa3.1 current through a Ca2+ and CaMKII-mediated pathway. (A) Summarized data showing ROS production by unstimulated MLS-9 microglial cells (control), and after treatment (20 min; 37°C) with db-cGMP (100 μM), with or without the PKG inhibitor, KT5823 (1 μM). Values are expressed as mean ± SEM (n = 5), and compared using a one-way ANOVA with Tukey’s post hoc test. ***p < 0.001 treatment versus control; †††p < 0.001 with and without PKG inhibitor. (B) Acute application of db-cGMP increases intracellular Ca2+ in MLS-9 cells but does not activate KCa3.1 current. Upper panel: Representative Fura-2 recording, in which 100 μM db-cGMP was bath applied during the period marked by the horizontal bar. The inset shows calibrated free intracellular Ca2+ concentration as mean ± SEM, n = 19 cells (***p < 0.001, Student’s t-test). Lower panel: Representative time course of current in a perforated-patch recording (same solutions and voltage protocols as Figure 1) in which db-cGMP (100 μM) was bath applied as indicated by the horizontal bar. (C) KCa3.1 current potentiation by db-cGMP is prevented by the ROS scavenger, MPG, and the CaMKII inhibitor, mAIP. Each set of three traces shows representative currents before and after adding 300 μM riluzole, with or without 1 μM TRAM-34. Upper panel: Representative recording from an MLS-9 cell pre-treated with 100 μM db-cGMP and the ROS scavenger, MPG (500 μM), for 20 min at room temperature. Lower panel: Cells were pre-treated with 100 μM db-cGMP and the CaM kinase II inhibitor, mAIP (1 μM) for 20 min at room temperature. (D) Summarized data from a population study with treatments as in panel C. The TRAM-34-sensitive KCa3.1 current is expressed as mean ± SEM for the number of cells indicated on each bar, and was compared using a one-way ANOVA with Tukey’s post hoc test; ***p < 0.001.

Mentions: The lack of effect of cGMP and PKG on excised patches (Figure 2) suggested that intracellular signaling was required for the current enhancement in MLS-9 cells (Figure 1). Therefore, we used perforated-patch recordings to maintain intracellular signaling when conducting experiments on native channels in MLS-9 cells and primary microglia. We first considered ROS because PKG increases ROS production in cardiac cells and neurons (28–30). Treating MLS-9 cells with db-cGMP increased ROS production by 38 ± 5% (Figure 3A; n = 5; p < 0.001), an effect that was prevented by the PKG inhibitor, KT5823. ROS can activate CaMKII (21, 47) and, as described in the Introduction, CaMK regulates native KCa3.1 channels in T lymphocytes (8). Intriguingly, CaMKII can be activated by ROS without elevated intracellular Ca2+ (21) or in a long-lasting Ca2+-independent manner after a transient Ca2+ elevation (47).


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)

PKG increases production of reactive oxygen species (ROS), which activates KCa3.1 current through a Ca2+ and CaMKII-mediated pathway. (A) Summarized data showing ROS production by unstimulated MLS-9 microglial cells (control), and after treatment (20 min; 37°C) with db-cGMP (100 μM), with or without the PKG inhibitor, KT5823 (1 μM). Values are expressed as mean ± SEM (n = 5), and compared using a one-way ANOVA with Tukey’s post hoc test. ***p < 0.001 treatment versus control; †††p < 0.001 with and without PKG inhibitor. (B) Acute application of db-cGMP increases intracellular Ca2+ in MLS-9 cells but does not activate KCa3.1 current. Upper panel: Representative Fura-2 recording, in which 100 μM db-cGMP was bath applied during the period marked by the horizontal bar. The inset shows calibrated free intracellular Ca2+ concentration as mean ± SEM, n = 19 cells (***p < 0.001, Student’s t-test). Lower panel: Representative time course of current in a perforated-patch recording (same solutions and voltage protocols as Figure 1) in which db-cGMP (100 μM) was bath applied as indicated by the horizontal bar. (C) KCa3.1 current potentiation by db-cGMP is prevented by the ROS scavenger, MPG, and the CaMKII inhibitor, mAIP. Each set of three traces shows representative currents before and after adding 300 μM riluzole, with or without 1 μM TRAM-34. Upper panel: Representative recording from an MLS-9 cell pre-treated with 100 μM db-cGMP and the ROS scavenger, MPG (500 μM), for 20 min at room temperature. Lower panel: Cells were pre-treated with 100 μM db-cGMP and the CaM kinase II inhibitor, mAIP (1 μM) for 20 min at room temperature. (D) Summarized data from a population study with treatments as in panel C. The TRAM-34-sensitive KCa3.1 current is expressed as mean ± SEM for the number of cells indicated on each bar, and was compared using a one-way ANOVA with Tukey’s post hoc test; ***p < 0.001.
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Related In: Results  -  Collection

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Figure 3: PKG increases production of reactive oxygen species (ROS), which activates KCa3.1 current through a Ca2+ and CaMKII-mediated pathway. (A) Summarized data showing ROS production by unstimulated MLS-9 microglial cells (control), and after treatment (20 min; 37°C) with db-cGMP (100 μM), with or without the PKG inhibitor, KT5823 (1 μM). Values are expressed as mean ± SEM (n = 5), and compared using a one-way ANOVA with Tukey’s post hoc test. ***p < 0.001 treatment versus control; †††p < 0.001 with and without PKG inhibitor. (B) Acute application of db-cGMP increases intracellular Ca2+ in MLS-9 cells but does not activate KCa3.1 current. Upper panel: Representative Fura-2 recording, in which 100 μM db-cGMP was bath applied during the period marked by the horizontal bar. The inset shows calibrated free intracellular Ca2+ concentration as mean ± SEM, n = 19 cells (***p < 0.001, Student’s t-test). Lower panel: Representative time course of current in a perforated-patch recording (same solutions and voltage protocols as Figure 1) in which db-cGMP (100 μM) was bath applied as indicated by the horizontal bar. (C) KCa3.1 current potentiation by db-cGMP is prevented by the ROS scavenger, MPG, and the CaMKII inhibitor, mAIP. Each set of three traces shows representative currents before and after adding 300 μM riluzole, with or without 1 μM TRAM-34. Upper panel: Representative recording from an MLS-9 cell pre-treated with 100 μM db-cGMP and the ROS scavenger, MPG (500 μM), for 20 min at room temperature. Lower panel: Cells were pre-treated with 100 μM db-cGMP and the CaM kinase II inhibitor, mAIP (1 μM) for 20 min at room temperature. (D) Summarized data from a population study with treatments as in panel C. The TRAM-34-sensitive KCa3.1 current is expressed as mean ± SEM for the number of cells indicated on each bar, and was compared using a one-way ANOVA with Tukey’s post hoc test; ***p < 0.001.
Mentions: The lack of effect of cGMP and PKG on excised patches (Figure 2) suggested that intracellular signaling was required for the current enhancement in MLS-9 cells (Figure 1). Therefore, we used perforated-patch recordings to maintain intracellular signaling when conducting experiments on native channels in MLS-9 cells and primary microglia. We first considered ROS because PKG increases ROS production in cardiac cells and neurons (28–30). Treating MLS-9 cells with db-cGMP increased ROS production by 38 ± 5% (Figure 3A; n = 5; p < 0.001), an effect that was prevented by the PKG inhibitor, KT5823. ROS can activate CaMKII (21, 47) and, as described in the Introduction, CaMK regulates native KCa3.1 channels in T lymphocytes (8). Intriguingly, CaMKII can be activated by ROS without elevated intracellular Ca2+ (21) or in a long-lasting Ca2+-independent manner after a transient Ca2+ elevation (47).

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