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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

H2O2 did not directly affect the KCa3.1 channels. (A) HEK293 cells transfected with hKCa3.1 were used to assess channel activity in inside-out patches in intracellular (bath) solutions containing 120 nM, 500 nM, or 1 μM free Ca2+ (as in Figure 2), with or without perfusing in 1 mM H2O2. Inward single-channel currents were recorded at −100 mV. (B) Summarized data show NPo in control bath solution, and 4–6 min after adding H2O2. Data are expressed as mean ± SEM from four patches per Ca2+ concentration, and a one-way ANOVA with Tukey’s post hoc test showed no differences following treatments.
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Figure 5: H2O2 did not directly affect the KCa3.1 channels. (A) HEK293 cells transfected with hKCa3.1 were used to assess channel activity in inside-out patches in intracellular (bath) solutions containing 120 nM, 500 nM, or 1 μM free Ca2+ (as in Figure 2), with or without perfusing in 1 mM H2O2. Inward single-channel currents were recorded at −100 mV. (B) Summarized data show NPo in control bath solution, and 4–6 min after adding H2O2. Data are expressed as mean ± SEM from four patches per Ca2+ concentration, and a one-way ANOVA with Tukey’s post hoc test showed no differences following treatments.

Mentions: Next, inside-out patches from transfected HEK293 cells were exploited to ask whether H2O2 can directly affect channel activity (NPo; calculated as in Figure 2). There were 1–3 active channels in each patch, channel activity increased with increasing Ca2+ (Figure 5), and the NPo values were the same as in Figure 2. Perfusing 1 mM H2O2 into the bath did not affect channel activity at any of the Ca2+ concentrations (Figure 5A, summarized in Figure 5B). At 120 nM Ca2+, NPo was 0.21 ± 0.05 (n = 4) and 0.23 ± 0.04 after adding H2O2. In 500 nM Ca2+, NPo was 1.17 ± 0.19 (n = 4) and 1.09 ± 0.21 after adding H2O2. At 1 μM Ca2+, NPo was 1.79 ± 0.26 (n = 4) and 1.88 ± 0.31 after adding H2O2. Thus, treatment with this ROS did not directly affect KCa3.1 activity, number of active channels, or their Ca2+ sensitivity; nor was the amplitude of single-channel currents affected (amplitude histograms; not shown). This supports the view that, in order to exert their modulatory effects on KCa3.1 channels, H2O2 and CaMKII require intact cells and possibly another mediator.


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)

H2O2 did not directly affect the KCa3.1 channels. (A) HEK293 cells transfected with hKCa3.1 were used to assess channel activity in inside-out patches in intracellular (bath) solutions containing 120 nM, 500 nM, or 1 μM free Ca2+ (as in Figure 2), with or without perfusing in 1 mM H2O2. Inward single-channel currents were recorded at −100 mV. (B) Summarized data show NPo in control bath solution, and 4–6 min after adding H2O2. Data are expressed as mean ± SEM from four patches per Ca2+ concentration, and a one-way ANOVA with Tukey’s post hoc test showed no differences following treatments.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 5: H2O2 did not directly affect the KCa3.1 channels. (A) HEK293 cells transfected with hKCa3.1 were used to assess channel activity in inside-out patches in intracellular (bath) solutions containing 120 nM, 500 nM, or 1 μM free Ca2+ (as in Figure 2), with or without perfusing in 1 mM H2O2. Inward single-channel currents were recorded at −100 mV. (B) Summarized data show NPo in control bath solution, and 4–6 min after adding H2O2. Data are expressed as mean ± SEM from four patches per Ca2+ concentration, and a one-way ANOVA with Tukey’s post hoc test showed no differences following treatments.
Mentions: Next, inside-out patches from transfected HEK293 cells were exploited to ask whether H2O2 can directly affect channel activity (NPo; calculated as in Figure 2). There were 1–3 active channels in each patch, channel activity increased with increasing Ca2+ (Figure 5), and the NPo values were the same as in Figure 2. Perfusing 1 mM H2O2 into the bath did not affect channel activity at any of the Ca2+ concentrations (Figure 5A, summarized in Figure 5B). At 120 nM Ca2+, NPo was 0.21 ± 0.05 (n = 4) and 0.23 ± 0.04 after adding H2O2. In 500 nM Ca2+, NPo was 1.17 ± 0.19 (n = 4) and 1.09 ± 0.21 after adding H2O2. At 1 μM Ca2+, NPo was 1.79 ± 0.26 (n = 4) and 1.88 ± 0.31 after adding H2O2. Thus, treatment with this ROS did not directly affect KCa3.1 activity, number of active channels, or their Ca2+ sensitivity; nor was the amplitude of single-channel currents affected (amplitude histograms; not shown). This supports the view that, in order to exert their modulatory effects on KCa3.1 channels, H2O2 and CaMKII require intact cells and possibly another mediator.

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