KCa3.1/IK1 Channel Regulation by cGMP-Dependent Protein Kinase (PKG) via Reactive Oxygen Species and CaMKII in Microglia: An Immune Modulating Feedback System?

Roger Ferreira,Lyanne C Schlichter,Raymond Wong

doi:10.3389/fimmu.2015.00153

Roger Ferreira, Lyanne C Schlichter + Show 1 more

Open Access

https://doi.org/10.3389/fimmu.2015.00153

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Abstract

The intermediate conductance Ca2+-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 Ca2+ 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 Ca2+, 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 (Po) or Ca2+ 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 Ca2+. 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.

Highlights

Following the discovery of a Ca2+-dependent K+ efflux (“Gardos” channel) in red blood cells [1], early patch-clamp studies focused on the intermediate-conductance Ca2+-dependent K+ (IK) channel in thymic T cells, B lymphocytes [2], and T cells [3, 4]
We found that KCa3.1 contributes to production of reactive oxygen species (ROS) [14], to p38 MAPK activation, nitric oxide, and peroxynitrite production in classical-activated microglia (“M1,” by analogy with macrophages polarized by T cells), and to their capacity to kill neurons in vitro and in vivo [15]
We recently discovered that when rat microglia are skewed to the anti-inflammatory“alternative” (M2) activation state using interleukin-4, both KCa3.1 expression and current are highly upregulated through the type I IL-4 receptor and subsequent signaling through JAK3, www.frontiersin.org

Summary

Introduction

Following the discovery of a Ca2+-dependent K+ efflux (“Gardos” channel) in red blood cells [1], early patch-clamp studies focused on the intermediate-conductance Ca2+-dependent K+ (IK) channel in thymic T cells, B lymphocytes [2], and T cells [3, 4]. After the KCNN4 gene was cloned [5,6,7] and identified as the IK channel ( called KCa3.1, IK1, IKCa1, SK4); its upregulation in activated T lymphocytes and crucial role in their proliferation [8, 9] generated interest in targeting this channel for immunosuppression [reviewed in Ref. KCa3.1 expression, activity, and contributions to T cell functions are governed by the cells’ activation state but until recently, little was known about this aspect for microglia. We recently discovered that when rat microglia are skewed to the anti-inflammatory“alternative” (M2) activation state using interleukin-4, both KCa3.1 expression and current are highly upregulated through the type I IL-4 receptor and subsequent signaling through JAK3, www.frontiersin.org

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