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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

Direct application of H2O2 increases the KCa3.1 current through a CaMKII-mediated pathway. (A) Hydrogen peroxide elevates intracellular Ca2+ but does not directly activate KCa3.1 current in MLS-9 cells. Upper panel: Representative Fura-2 recording, in which 1 mM H2O2 was bath applied during the period marked by the horizontal bar. The inset shows calibrated free intracellular Ca2+ concentration as mean ± SEM, n = 18 cells (***p < 0.001, Student’s t-test). Lower panel: Representative current in a perforated-patch recording (same solutions and voltage protocols as Figure 1) with 1 mM H2O2 bath applied as indicated. (B) Representative KCa3.1 current traces in perforated-patch recordings, representative currents before and after adding 300 μM riluzole, with or without 1 μM TRAM-34. From top to bottom: control cell, cell pre-treated with 1 mM H2O2 for 10 min at room temperature, cell pre-treated with both 1 mM H2O2 and the ROS scavenger, MPG (500 μM; 10 min, room temperature), cell pre-treated with 1 mM H2O2, and the CaMKII inhibitor, mAIP (1 μM; 10 min, room temperature). (C) Summarized data from a population study with treatments as in panel (B). 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 < 0.0001.
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Figure 4: Direct application of H2O2 increases the KCa3.1 current through a CaMKII-mediated pathway. (A) Hydrogen peroxide elevates intracellular Ca2+ but does not directly activate KCa3.1 current in MLS-9 cells. Upper panel: Representative Fura-2 recording, in which 1 mM H2O2 was bath applied during the period marked by the horizontal bar. The inset shows calibrated free intracellular Ca2+ concentration as mean ± SEM, n = 18 cells (***p < 0.001, Student’s t-test). Lower panel: Representative current in a perforated-patch recording (same solutions and voltage protocols as Figure 1) with 1 mM H2O2 bath applied as indicated. (B) Representative KCa3.1 current traces in perforated-patch recordings, representative currents before and after adding 300 μM riluzole, with or without 1 μM TRAM-34. From top to bottom: control cell, cell pre-treated with 1 mM H2O2 for 10 min at room temperature, cell pre-treated with both 1 mM H2O2 and the ROS scavenger, MPG (500 μM; 10 min, room temperature), cell pre-treated with 1 mM H2O2, and the CaMKII inhibitor, mAIP (1 μM; 10 min, room temperature). (C) Summarized data from a population study with treatments as in panel (B). 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 < 0.0001.

Mentions: To further analyze the ROS-mediated increase in KCa3.1 current, we tested acute application of the relatively stable hydrogen peroxide (1 mM H2O2) molecule. Treating MLS-9 cells with H2O2 evoked a moderate, transient rise in intracellular Ca2+ (Figure 4A) that peaked at 456 ± 33 nM (n = 18) by 11.6 ± 0.1 min after treatment. The peak elevation in Ca2+ evoked by H2O2 was higher than for db-cGMP and occurred ~5 min sooner. Again, it was insufficient to activate the KCa3.1 current in MLS-9 cells (n = 6 cells tested; example in Figure 4A), which requires supra-micromolar concentrations (described above). In contrast, in perforated-patch recordings from cells that were pre-incubated with 1 mM H2O2, the KCa3.1 current was more than twofold larger (21.5 ± 1.6 pA/pF; n = 6; p < 0.0001) than in control cells (10.3 ± 0.5 pA/pF; n = 4; Figures 4B,C). The potentiation of the current was prevented if cells were simultaneously pre-treated with the ROS scavenger (500 μM MGP) or the CaMKII inhibitor (1 μM mAIP). The current density remained at 9.5 ± 1.4 pA/pF in MGP-treated cells and 9.2 ± 1.8 pA/pF in mAIP-treated cells. These data show that KCa3.1 function can be enhanced by this identified, stable ROS species through a similar pathway involving CaMKII.


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)

Direct application of H2O2 increases the KCa3.1 current through a CaMKII-mediated pathway. (A) Hydrogen peroxide elevates intracellular Ca2+ but does not directly activate KCa3.1 current in MLS-9 cells. Upper panel: Representative Fura-2 recording, in which 1 mM H2O2 was bath applied during the period marked by the horizontal bar. The inset shows calibrated free intracellular Ca2+ concentration as mean ± SEM, n = 18 cells (***p < 0.001, Student’s t-test). Lower panel: Representative current in a perforated-patch recording (same solutions and voltage protocols as Figure 1) with 1 mM H2O2 bath applied as indicated. (B) Representative KCa3.1 current traces in perforated-patch recordings, representative currents before and after adding 300 μM riluzole, with or without 1 μM TRAM-34. From top to bottom: control cell, cell pre-treated with 1 mM H2O2 for 10 min at room temperature, cell pre-treated with both 1 mM H2O2 and the ROS scavenger, MPG (500 μM; 10 min, room temperature), cell pre-treated with 1 mM H2O2, and the CaMKII inhibitor, mAIP (1 μM; 10 min, room temperature). (C) Summarized data from a population study with treatments as in panel (B). 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 < 0.0001.
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Related In: Results  -  Collection

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Figure 4: Direct application of H2O2 increases the KCa3.1 current through a CaMKII-mediated pathway. (A) Hydrogen peroxide elevates intracellular Ca2+ but does not directly activate KCa3.1 current in MLS-9 cells. Upper panel: Representative Fura-2 recording, in which 1 mM H2O2 was bath applied during the period marked by the horizontal bar. The inset shows calibrated free intracellular Ca2+ concentration as mean ± SEM, n = 18 cells (***p < 0.001, Student’s t-test). Lower panel: Representative current in a perforated-patch recording (same solutions and voltage protocols as Figure 1) with 1 mM H2O2 bath applied as indicated. (B) Representative KCa3.1 current traces in perforated-patch recordings, representative currents before and after adding 300 μM riluzole, with or without 1 μM TRAM-34. From top to bottom: control cell, cell pre-treated with 1 mM H2O2 for 10 min at room temperature, cell pre-treated with both 1 mM H2O2 and the ROS scavenger, MPG (500 μM; 10 min, room temperature), cell pre-treated with 1 mM H2O2, and the CaMKII inhibitor, mAIP (1 μM; 10 min, room temperature). (C) Summarized data from a population study with treatments as in panel (B). 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 < 0.0001.
Mentions: To further analyze the ROS-mediated increase in KCa3.1 current, we tested acute application of the relatively stable hydrogen peroxide (1 mM H2O2) molecule. Treating MLS-9 cells with H2O2 evoked a moderate, transient rise in intracellular Ca2+ (Figure 4A) that peaked at 456 ± 33 nM (n = 18) by 11.6 ± 0.1 min after treatment. The peak elevation in Ca2+ evoked by H2O2 was higher than for db-cGMP and occurred ~5 min sooner. Again, it was insufficient to activate the KCa3.1 current in MLS-9 cells (n = 6 cells tested; example in Figure 4A), which requires supra-micromolar concentrations (described above). In contrast, in perforated-patch recordings from cells that were pre-incubated with 1 mM H2O2, the KCa3.1 current was more than twofold larger (21.5 ± 1.6 pA/pF; n = 6; p < 0.0001) than in control cells (10.3 ± 0.5 pA/pF; n = 4; Figures 4B,C). The potentiation of the current was prevented if cells were simultaneously pre-treated with the ROS scavenger (500 μM MGP) or the CaMKII inhibitor (1 μM mAIP). The current density remained at 9.5 ± 1.4 pA/pF in MGP-treated cells and 9.2 ± 1.8 pA/pF in mAIP-treated cells. These data show that KCa3.1 function can be enhanced by this identified, stable ROS species through a similar pathway involving CaMKII.

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