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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

The endogenous KCa3.1 current in MLS-9 microglial cells is increased by cGMP, which requires cGMP-protein kinase. For all traces, the voltage protocol was a holding potential of –70 mV, and repeated ramps from –100 to +80 mV. Recordings were conducted at room temperature in the perforated-patch configuration, and riluzole was used simply to activate the KCa3.1 current at the normal low intracellular Ca2+ concentration. The bath always contained 100 nM apamin, a KCa2.1–2.3 channel blocker. (A)Upper: Representative current traces from a control cell (trace marked “ctrl”), followed by bath addition of 300 μM riluzole, and then 1 μM of the selective KCa3.1 blocker, TRAM-34. Lower: The time course of current activation and block by 1 μM TRAM-34. (B,C) Representative current traces from cells before and after activating the current with riluzole; with or without 1 μM TRAM-34. Cells were pre-treated with the membrane-permeant cGMP analog, db-cGMP (100 μM), for 20 min at room temperature, without (B) or with (C) 1 μM KT5823, a selective inhibitor of cGMP-protein kinase (PKG). (D) Summarized data from a population study using the treatments in panels A–C. For each cell, the KCa3.1 current amplitude was measured at +80 mV, as the component of the riluzole-activated current that was blocked by TRAM-34 (1 μM). The current was always normalized to the cell capacitance (in pF) to account for any differences in cell size and expressed as current density. The TRAM-34-sensitive KCa3.1 current is expressed as mean ± SEM for the number of cells indicated on each bar, and data were compared using one-way ANOVA, with Tukey’s post hoc test. **p < 0.01, indicates a difference from both controls and KT5823-treated cells. ††p < 0.01, for the comparison indicated. There was no difference between the control and KT5823-treated cells.
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Figure 1: The endogenous KCa3.1 current in MLS-9 microglial cells is increased by cGMP, which requires cGMP-protein kinase. For all traces, the voltage protocol was a holding potential of –70 mV, and repeated ramps from –100 to +80 mV. Recordings were conducted at room temperature in the perforated-patch configuration, and riluzole was used simply to activate the KCa3.1 current at the normal low intracellular Ca2+ concentration. The bath always contained 100 nM apamin, a KCa2.1–2.3 channel blocker. (A)Upper: Representative current traces from a control cell (trace marked “ctrl”), followed by bath addition of 300 μM riluzole, and then 1 μM of the selective KCa3.1 blocker, TRAM-34. Lower: The time course of current activation and block by 1 μM TRAM-34. (B,C) Representative current traces from cells before and after activating the current with riluzole; with or without 1 μM TRAM-34. Cells were pre-treated with the membrane-permeant cGMP analog, db-cGMP (100 μM), for 20 min at room temperature, without (B) or with (C) 1 μM KT5823, a selective inhibitor of cGMP-protein kinase (PKG). (D) Summarized data from a population study using the treatments in panels A–C. For each cell, the KCa3.1 current amplitude was measured at +80 mV, as the component of the riluzole-activated current that was blocked by TRAM-34 (1 μM). The current was always normalized to the cell capacitance (in pF) to account for any differences in cell size and expressed as current density. The TRAM-34-sensitive KCa3.1 current is expressed as mean ± SEM for the number of cells indicated on each bar, and data were compared using one-way ANOVA, with Tukey’s post hoc test. **p < 0.01, indicates a difference from both controls and KT5823-treated cells. ††p < 0.01, for the comparison indicated. There was no difference between the control and KT5823-treated cells.

Mentions: To study the native channels in MLS-9 and primary microglial cells, we used riluzole because it reliably activated a KCa3.1 current in perforated-patch recordings. The current was also stable enough to add TRAM-34 to confirm the channel identity and quantify the current density (20, 31). As expected, the KCa3.1 current was not activated at resting levels of intracellular Ca2+ in MLS-9 cells. However, a stable KCa3.1 current was activated by riluzole in all cells tested (Figure 1A). As expected for KCa3.1, current activation was independent of voltage, and it reversed close to the Nernst potential for K+ (–84 mV with the solutions used). The current was entirely KCa3.1 (in the presence of apamin), as demonstrated by full inhibition by the selective KCa3.1 blocker, 1 μM TRAM-34 (Figure 1A). In all subsequent experiments, the KCa3.1 current was quantified as the TRAM-34-sensitive component.


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)

The endogenous KCa3.1 current in MLS-9 microglial cells is increased by cGMP, which requires cGMP-protein kinase. For all traces, the voltage protocol was a holding potential of –70 mV, and repeated ramps from –100 to +80 mV. Recordings were conducted at room temperature in the perforated-patch configuration, and riluzole was used simply to activate the KCa3.1 current at the normal low intracellular Ca2+ concentration. The bath always contained 100 nM apamin, a KCa2.1–2.3 channel blocker. (A)Upper: Representative current traces from a control cell (trace marked “ctrl”), followed by bath addition of 300 μM riluzole, and then 1 μM of the selective KCa3.1 blocker, TRAM-34. Lower: The time course of current activation and block by 1 μM TRAM-34. (B,C) Representative current traces from cells before and after activating the current with riluzole; with or without 1 μM TRAM-34. Cells were pre-treated with the membrane-permeant cGMP analog, db-cGMP (100 μM), for 20 min at room temperature, without (B) or with (C) 1 μM KT5823, a selective inhibitor of cGMP-protein kinase (PKG). (D) Summarized data from a population study using the treatments in panels A–C. For each cell, the KCa3.1 current amplitude was measured at +80 mV, as the component of the riluzole-activated current that was blocked by TRAM-34 (1 μM). The current was always normalized to the cell capacitance (in pF) to account for any differences in cell size and expressed as current density. The TRAM-34-sensitive KCa3.1 current is expressed as mean ± SEM for the number of cells indicated on each bar, and data were compared using one-way ANOVA, with Tukey’s post hoc test. **p < 0.01, indicates a difference from both controls and KT5823-treated cells. ††p < 0.01, for the comparison indicated. There was no difference between the control and KT5823-treated cells.
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Related In: Results  -  Collection

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Figure 1: The endogenous KCa3.1 current in MLS-9 microglial cells is increased by cGMP, which requires cGMP-protein kinase. For all traces, the voltage protocol was a holding potential of –70 mV, and repeated ramps from –100 to +80 mV. Recordings were conducted at room temperature in the perforated-patch configuration, and riluzole was used simply to activate the KCa3.1 current at the normal low intracellular Ca2+ concentration. The bath always contained 100 nM apamin, a KCa2.1–2.3 channel blocker. (A)Upper: Representative current traces from a control cell (trace marked “ctrl”), followed by bath addition of 300 μM riluzole, and then 1 μM of the selective KCa3.1 blocker, TRAM-34. Lower: The time course of current activation and block by 1 μM TRAM-34. (B,C) Representative current traces from cells before and after activating the current with riluzole; with or without 1 μM TRAM-34. Cells were pre-treated with the membrane-permeant cGMP analog, db-cGMP (100 μM), for 20 min at room temperature, without (B) or with (C) 1 μM KT5823, a selective inhibitor of cGMP-protein kinase (PKG). (D) Summarized data from a population study using the treatments in panels A–C. For each cell, the KCa3.1 current amplitude was measured at +80 mV, as the component of the riluzole-activated current that was blocked by TRAM-34 (1 μM). The current was always normalized to the cell capacitance (in pF) to account for any differences in cell size and expressed as current density. The TRAM-34-sensitive KCa3.1 current is expressed as mean ± SEM for the number of cells indicated on each bar, and data were compared using one-way ANOVA, with Tukey’s post hoc test. **p < 0.01, indicates a difference from both controls and KT5823-treated cells. ††p < 0.01, for the comparison indicated. There was no difference between the control and KT5823-treated cells.
Mentions: To study the native channels in MLS-9 and primary microglial cells, we used riluzole because it reliably activated a KCa3.1 current in perforated-patch recordings. The current was also stable enough to add TRAM-34 to confirm the channel identity and quantify the current density (20, 31). As expected, the KCa3.1 current was not activated at resting levels of intracellular Ca2+ in MLS-9 cells. However, a stable KCa3.1 current was activated by riluzole in all cells tested (Figure 1A). As expected for KCa3.1, current activation was independent of voltage, and it reversed close to the Nernst potential for K+ (–84 mV with the solutions used). The current was entirely KCa3.1 (in the presence of apamin), as demonstrated by full inhibition by the selective KCa3.1 blocker, 1 μM TRAM-34 (Figure 1A). In all subsequent experiments, the KCa3.1 current was quantified as the TRAM-34-sensitive component.

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