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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

In alternative-activated rat microglia, ROS production requires and potentiates KCa3.1 channel activity through the ROS-PKG-CaMKII pathway. To evoke alternative activation, primary rat microglia were treated with rat recombinant interleukin-4 (IL-4); 20 ng/mL for 24 or 48 h. (A) Summarized data show ROS production, detected by CM-H2DCFDA (see Methods) in untreated versus IL-4 treated microglia. Under each condition, separate batches of microglia were exposed to the PKG inhibitor (1 μM KT5823) or the KCa3.1 blocker (1 μM TRAM-34) for 24 or 48 h at 37°C. Values are expressed as mean ± SEM (n = 6 replicates experiments each), and compared using a two-way ANOVA with Tukey’s post hoc test. **p < 0.01 and ***p < 0.001 for non-activated versus IL-4 treated cells; #p < 0.05 and ††p < 0.01, for drug treatments, as indicated. (B) KCa3.1 currents were recorded in alternative-activated microglia 48 h after IL-4 treatment, using the perforated-patch configuration and the same solutions and voltage protocols as in Figure 1. (i) Representative currents from a cell before and after adding the KCa3.1 activator, 300 μM riluzole, and the KCa3.1 blocker, 1 μM TRAM-34. (ii) A cell pre-treated with 100 μM db-cGMP for 20 min at room temperature. (iii) Summarized data from a population study, in which the TRAM-34-sensitive KCa3.1 current amplitude is expressed as mean ± SEM (n = 4 cells each). The difference was non-significant based on Student’s t-test. (C). KCa3.1 currents were recorded from alternative-activated microglia, with and without the activator, riluzole, as in panel B. All drug pre-treatments were for 1 h at 37°C. From top to bottom, different microglia were treated with 1 μM KT5823; the ROS scavenger, 500 μM MPG; the CaMKII inhibitor, 1 μM mAIP; KT5823, MPG and mAIP. (D). Summarized data from a population study of experiments as in panel (C). The TRAM-34-sensitive KCa3.1 current is expressed as the mean ± SEM (n = 5 cells each), and was compared using a one-way ANOVA with Tukey’s post hoc test; *p < 0.05, **p < 0.01, ***p < 0.001.
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Figure 6: In alternative-activated rat microglia, ROS production requires and potentiates KCa3.1 channel activity through the ROS-PKG-CaMKII pathway. To evoke alternative activation, primary rat microglia were treated with rat recombinant interleukin-4 (IL-4); 20 ng/mL for 24 or 48 h. (A) Summarized data show ROS production, detected by CM-H2DCFDA (see Methods) in untreated versus IL-4 treated microglia. Under each condition, separate batches of microglia were exposed to the PKG inhibitor (1 μM KT5823) or the KCa3.1 blocker (1 μM TRAM-34) for 24 or 48 h at 37°C. Values are expressed as mean ± SEM (n = 6 replicates experiments each), and compared using a two-way ANOVA with Tukey’s post hoc test. **p < 0.01 and ***p < 0.001 for non-activated versus IL-4 treated cells; #p < 0.05 and ††p < 0.01, for drug treatments, as indicated. (B) KCa3.1 currents were recorded in alternative-activated microglia 48 h after IL-4 treatment, using the perforated-patch configuration and the same solutions and voltage protocols as in Figure 1. (i) Representative currents from a cell before and after adding the KCa3.1 activator, 300 μM riluzole, and the KCa3.1 blocker, 1 μM TRAM-34. (ii) A cell pre-treated with 100 μM db-cGMP for 20 min at room temperature. (iii) Summarized data from a population study, in which the TRAM-34-sensitive KCa3.1 current amplitude is expressed as mean ± SEM (n = 4 cells each). The difference was non-significant based on Student’s t-test. (C). KCa3.1 currents were recorded from alternative-activated microglia, with and without the activator, riluzole, as in panel B. All drug pre-treatments were for 1 h at 37°C. From top to bottom, different microglia were treated with 1 μM KT5823; the ROS scavenger, 500 μM MPG; the CaMKII inhibitor, 1 μM mAIP; KT5823, MPG and mAIP. (D). Summarized data from a population study of experiments as in panel (C). The TRAM-34-sensitive KCa3.1 current is expressed as the mean ± SEM (n = 5 cells each), and was compared using a one-way ANOVA with Tukey’s post hoc test; *p < 0.05, **p < 0.01, ***p < 0.001.

Mentions: The above results on MLS-9 cells show that ROS and cGMP increase KCa3.1 current through a pathway requiring PKG and CaMKII. It was important to determine whether the same pathway regulates KCa3.1 in primary cultured microglia. For these experiments, we used alternative-activated (IL-4 treated) rat microglia, which have upregulated KCNN4 expression and a much larger KCa3.1 current than resting microglia (20), and in which KCa3.1 contributes to Ca2+ signaling (27) and migration (20). Here, we found that at 24 and 48 h after IL-4 treatment, ROS production was increased by 93 ± 31% (p < 0.01) and 78 ± 21% (p < 0.01), respectively (Figure 6A). The PKG inhibitor, KT5823, decreased this induced ROS production by 53% at 24 h and 73% at 48 h after IL-4 treatment, but did not significantly affect resting microglia. The enhanced ROS production in alternative-activated microglia was moderately dependent on KCa3.1 channels, as TRAM-34 reduced it by 29% at 24 h and 31% at 48 h after IL-4 treatment.


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)

In alternative-activated rat microglia, ROS production requires and potentiates KCa3.1 channel activity through the ROS-PKG-CaMKII pathway. To evoke alternative activation, primary rat microglia were treated with rat recombinant interleukin-4 (IL-4); 20 ng/mL for 24 or 48 h. (A) Summarized data show ROS production, detected by CM-H2DCFDA (see Methods) in untreated versus IL-4 treated microglia. Under each condition, separate batches of microglia were exposed to the PKG inhibitor (1 μM KT5823) or the KCa3.1 blocker (1 μM TRAM-34) for 24 or 48 h at 37°C. Values are expressed as mean ± SEM (n = 6 replicates experiments each), and compared using a two-way ANOVA with Tukey’s post hoc test. **p < 0.01 and ***p < 0.001 for non-activated versus IL-4 treated cells; #p < 0.05 and ††p < 0.01, for drug treatments, as indicated. (B) KCa3.1 currents were recorded in alternative-activated microglia 48 h after IL-4 treatment, using the perforated-patch configuration and the same solutions and voltage protocols as in Figure 1. (i) Representative currents from a cell before and after adding the KCa3.1 activator, 300 μM riluzole, and the KCa3.1 blocker, 1 μM TRAM-34. (ii) A cell pre-treated with 100 μM db-cGMP for 20 min at room temperature. (iii) Summarized data from a population study, in which the TRAM-34-sensitive KCa3.1 current amplitude is expressed as mean ± SEM (n = 4 cells each). The difference was non-significant based on Student’s t-test. (C). KCa3.1 currents were recorded from alternative-activated microglia, with and without the activator, riluzole, as in panel B. All drug pre-treatments were for 1 h at 37°C. From top to bottom, different microglia were treated with 1 μM KT5823; the ROS scavenger, 500 μM MPG; the CaMKII inhibitor, 1 μM mAIP; KT5823, MPG and mAIP. (D). Summarized data from a population study of experiments as in panel (C). The TRAM-34-sensitive KCa3.1 current is expressed as the mean ± SEM (n = 5 cells each), and was compared using a one-way ANOVA with Tukey’s post hoc test; *p < 0.05, **p < 0.01, ***p < 0.001.
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Figure 6: In alternative-activated rat microglia, ROS production requires and potentiates KCa3.1 channel activity through the ROS-PKG-CaMKII pathway. To evoke alternative activation, primary rat microglia were treated with rat recombinant interleukin-4 (IL-4); 20 ng/mL for 24 or 48 h. (A) Summarized data show ROS production, detected by CM-H2DCFDA (see Methods) in untreated versus IL-4 treated microglia. Under each condition, separate batches of microglia were exposed to the PKG inhibitor (1 μM KT5823) or the KCa3.1 blocker (1 μM TRAM-34) for 24 or 48 h at 37°C. Values are expressed as mean ± SEM (n = 6 replicates experiments each), and compared using a two-way ANOVA with Tukey’s post hoc test. **p < 0.01 and ***p < 0.001 for non-activated versus IL-4 treated cells; #p < 0.05 and ††p < 0.01, for drug treatments, as indicated. (B) KCa3.1 currents were recorded in alternative-activated microglia 48 h after IL-4 treatment, using the perforated-patch configuration and the same solutions and voltage protocols as in Figure 1. (i) Representative currents from a cell before and after adding the KCa3.1 activator, 300 μM riluzole, and the KCa3.1 blocker, 1 μM TRAM-34. (ii) A cell pre-treated with 100 μM db-cGMP for 20 min at room temperature. (iii) Summarized data from a population study, in which the TRAM-34-sensitive KCa3.1 current amplitude is expressed as mean ± SEM (n = 4 cells each). The difference was non-significant based on Student’s t-test. (C). KCa3.1 currents were recorded from alternative-activated microglia, with and without the activator, riluzole, as in panel B. All drug pre-treatments were for 1 h at 37°C. From top to bottom, different microglia were treated with 1 μM KT5823; the ROS scavenger, 500 μM MPG; the CaMKII inhibitor, 1 μM mAIP; KT5823, MPG and mAIP. (D). Summarized data from a population study of experiments as in panel (C). The TRAM-34-sensitive KCa3.1 current is expressed as the mean ± SEM (n = 5 cells each), and was compared using a one-way ANOVA with Tukey’s post hoc test; *p < 0.05, **p < 0.01, ***p < 0.001.
Mentions: The above results on MLS-9 cells show that ROS and cGMP increase KCa3.1 current through a pathway requiring PKG and CaMKII. It was important to determine whether the same pathway regulates KCa3.1 in primary cultured microglia. For these experiments, we used alternative-activated (IL-4 treated) rat microglia, which have upregulated KCNN4 expression and a much larger KCa3.1 current than resting microglia (20), and in which KCa3.1 contributes to Ca2+ signaling (27) and migration (20). Here, we found that at 24 and 48 h after IL-4 treatment, ROS production was increased by 93 ± 31% (p < 0.01) and 78 ± 21% (p < 0.01), respectively (Figure 6A). The PKG inhibitor, KT5823, decreased this induced ROS production by 53% at 24 h and 73% at 48 h after IL-4 treatment, but did not significantly affect resting microglia. The enhanced ROS production in alternative-activated microglia was moderately dependent on KCa3.1 channels, as TRAM-34 reduced it by 29% at 24 h and 31% at 48 h after IL-4 treatment.

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