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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

cGMP-protein kinase (PKG) did not directly affect KCa3.1 channel activity. Inside-out patches were excised from HEK293 cells that had been transfected with wild-type human KCNN4 (KCa3.1). The bath and pipette solutions both contained 140 mM potassium, and unless otherwise indicated, inward single-channel currents were recorded at a membrane potential of −100 mV. (A) KCa3.1 channel activity was recorded with intracellular solutions containing ATP and 120 nM, 500 nM, or 1 μM free Ca2+, sequentially perfused into the bath. At the end of the recording, the KCa3.1 selective blocker, 1 μM TRAM-34, was perfused in. Patches usually contained multiple channels, and the dashes indicate the closed level and opening of 1, 2, or 3 channels. (B–D) At each Ca2+ concentration (120 nM, 500 nM, 1 μM), the bath was sequentially perfused with cGMP (100 μM), and cGMP + PKG holoenzyme (1 U/μL). (E) Summarized data show NPo in control bath solution and 4–6 min after adding cGMP or cGMP + PKG. Data are expressed as mean ± SEM for the number of patches indicated on the bars. The dashed lines indicate the NPo value in control bath solution at each Ca2+ concentration. A two-way ANOVA with Tukey’s post hoc test shows that activity increased with intracellular Ca2+ (**p < 0.01 for 500 nM Ca2+, and ***p < 0.001 for 1 μM Ca2+) and was significantly reduced by 1 μM TRAM-34 (only data for 1 μM Ca2+ shown; ###p < 0.001). There were no differences with cGMP or cGMP/PKG treatments.
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Figure 2: cGMP-protein kinase (PKG) did not directly affect KCa3.1 channel activity. Inside-out patches were excised from HEK293 cells that had been transfected with wild-type human KCNN4 (KCa3.1). The bath and pipette solutions both contained 140 mM potassium, and unless otherwise indicated, inward single-channel currents were recorded at a membrane potential of −100 mV. (A) KCa3.1 channel activity was recorded with intracellular solutions containing ATP and 120 nM, 500 nM, or 1 μM free Ca2+, sequentially perfused into the bath. At the end of the recording, the KCa3.1 selective blocker, 1 μM TRAM-34, was perfused in. Patches usually contained multiple channels, and the dashes indicate the closed level and opening of 1, 2, or 3 channels. (B–D) At each Ca2+ concentration (120 nM, 500 nM, 1 μM), the bath was sequentially perfused with cGMP (100 μM), and cGMP + PKG holoenzyme (1 U/μL). (E) Summarized data show NPo in control bath solution and 4–6 min after adding cGMP or cGMP + PKG. Data are expressed as mean ± SEM for the number of patches indicated on the bars. The dashed lines indicate the NPo value in control bath solution at each Ca2+ concentration. A two-way ANOVA with Tukey’s post hoc test shows that activity increased with intracellular Ca2+ (**p < 0.01 for 500 nM Ca2+, and ***p < 0.001 for 1 μM Ca2+) and was significantly reduced by 1 μM TRAM-34 (only data for 1 μM Ca2+ shown; ###p < 0.001). There were no differences with cGMP or cGMP/PKG treatments.

Mentions: Inside-out patches were excised into a bath (intracellular) solution containing 100 μM ATP and free Ca2+ concentrations of 120 nM, 500 nM, or 1 μM (Figure 2). The intracellular and extracellular solutions contained symmetrical high K+ (140 mM) to set the Nernst potential to 0 mV, increase the unitary inward current amplitude, and expose the innate inward rectification of the single-channel current. One to three channels were usually active in each patch, their activity was stable for several minutes, and channel activity was recorded at –100 mV. Channel activity was quantified as NPo: the number of active channels, N, times the open probability, Po. Using representative 2 min-long segments of each recording, thresholds were set for the closed level and each open level (based on amplitude histograms; see Methods).


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)

cGMP-protein kinase (PKG) did not directly affect KCa3.1 channel activity. Inside-out patches were excised from HEK293 cells that had been transfected with wild-type human KCNN4 (KCa3.1). The bath and pipette solutions both contained 140 mM potassium, and unless otherwise indicated, inward single-channel currents were recorded at a membrane potential of −100 mV. (A) KCa3.1 channel activity was recorded with intracellular solutions containing ATP and 120 nM, 500 nM, or 1 μM free Ca2+, sequentially perfused into the bath. At the end of the recording, the KCa3.1 selective blocker, 1 μM TRAM-34, was perfused in. Patches usually contained multiple channels, and the dashes indicate the closed level and opening of 1, 2, or 3 channels. (B–D) At each Ca2+ concentration (120 nM, 500 nM, 1 μM), the bath was sequentially perfused with cGMP (100 μM), and cGMP + PKG holoenzyme (1 U/μL). (E) Summarized data show NPo in control bath solution and 4–6 min after adding cGMP or cGMP + PKG. Data are expressed as mean ± SEM for the number of patches indicated on the bars. The dashed lines indicate the NPo value in control bath solution at each Ca2+ concentration. A two-way ANOVA with Tukey’s post hoc test shows that activity increased with intracellular Ca2+ (**p < 0.01 for 500 nM Ca2+, and ***p < 0.001 for 1 μM Ca2+) and was significantly reduced by 1 μM TRAM-34 (only data for 1 μM Ca2+ shown; ###p < 0.001). There were no differences with cGMP or cGMP/PKG treatments.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: cGMP-protein kinase (PKG) did not directly affect KCa3.1 channel activity. Inside-out patches were excised from HEK293 cells that had been transfected with wild-type human KCNN4 (KCa3.1). The bath and pipette solutions both contained 140 mM potassium, and unless otherwise indicated, inward single-channel currents were recorded at a membrane potential of −100 mV. (A) KCa3.1 channel activity was recorded with intracellular solutions containing ATP and 120 nM, 500 nM, or 1 μM free Ca2+, sequentially perfused into the bath. At the end of the recording, the KCa3.1 selective blocker, 1 μM TRAM-34, was perfused in. Patches usually contained multiple channels, and the dashes indicate the closed level and opening of 1, 2, or 3 channels. (B–D) At each Ca2+ concentration (120 nM, 500 nM, 1 μM), the bath was sequentially perfused with cGMP (100 μM), and cGMP + PKG holoenzyme (1 U/μL). (E) Summarized data show NPo in control bath solution and 4–6 min after adding cGMP or cGMP + PKG. Data are expressed as mean ± SEM for the number of patches indicated on the bars. The dashed lines indicate the NPo value in control bath solution at each Ca2+ concentration. A two-way ANOVA with Tukey’s post hoc test shows that activity increased with intracellular Ca2+ (**p < 0.01 for 500 nM Ca2+, and ***p < 0.001 for 1 μM Ca2+) and was significantly reduced by 1 μM TRAM-34 (only data for 1 μM Ca2+ shown; ###p < 0.001). There were no differences with cGMP or cGMP/PKG treatments.
Mentions: Inside-out patches were excised into a bath (intracellular) solution containing 100 μM ATP and free Ca2+ concentrations of 120 nM, 500 nM, or 1 μM (Figure 2). The intracellular and extracellular solutions contained symmetrical high K+ (140 mM) to set the Nernst potential to 0 mV, increase the unitary inward current amplitude, and expose the innate inward rectification of the single-channel current. One to three channels were usually active in each patch, their activity was stable for several minutes, and channel activity was recorded at –100 mV. Channel activity was quantified as NPo: the number of active channels, N, times the open probability, Po. Using representative 2 min-long segments of each recording, thresholds were set for the closed level and each open level (based on amplitude histograms; see Methods).

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