Expression and contributions of the Kir2.1 inward-rectifier K(+) channel to proliferation, migration and chemotaxis of microglia in unstimulated and anti-inflammatory states.

Doris Lam,Lyanne C Schlichter

doi:10.3389/fncel.2015.00185

Abstract

When microglia respond to CNS damage, they can range from pro-inflammatory (classical, M1) to anti-inflammatory, alternative (M2) and acquired deactivation states. It is important to determine how microglial functions are affected by these activation states, and to identify molecules that regulate their behavior. Microglial proliferation and migration are crucial during development and following damage in the adult, and both functions are Ca2+-dependent. In many cell types, the membrane potential and driving force for Ca2+ influx are regulated by inward-rectifier K+ channels, including Kir2.1, which is prevalent in microglia. However, it is not known whether Kir2.1 expression and contributions are altered in anti-inflammatory states. We tested the hypothesis that Kir2.1 contributes to Ca2+ entry, proliferation and migration of rat microglia. Kir2.1 (KCNJ2) transcript expression, current amplitude, and proliferation were comparable in unstimulated microglia and following alternative activation (IL-4 stimulated) and acquired deactivation (IL-10 stimulated). To examine functional roles of Kir2.1 in microglia, we first determined that ML133 was more effective than the commonly used blocker, Ba2+; i.e., ML133 was potent (IC50 = 3.5 μM) and voltage independent. Both blockers slightly increased proliferation in unstimulated or IL-4 (but not IL-10)-stimulated microglia. Stimulation with IL-4 or IL-10 increased migration and ATP-induced chemotaxis, and blocking Kir2.1 greatly reduced both but ML133 was more effective. In all three activation states, blocking Kir2.1 with ML133 dramatically reduced Ca2+ influx through Ca2+-release-activated Ca2+ (CRAC) channels. Thus, Kir2.1 channel activity is necessary for microglial Ca2+ signaling and migration under resting and anti-inflammatory states but the channel weakly inhibits proliferation.

Highlights

Members of the Kir2 inward-rectifier K+ channel family are expressed in both excitable and nonexcitable cells, where their primary function is to maintain a hyperpolarized membrane potential (Lu, 2004; Hibino et al, 2010)
Kir2.1 Expression and Current in Anti-Inflammatory Microglial Activation States We previously found that alternative activation and acquired deactivation are elicited by 24 h stimulation of rat microglia with IL-4 or IL-10, respectively (Lively and Schlichter, 2013; Ferreira et al, 2014; Siddiqui et al, 2014)
Kir2.1 Contributes to Microglial Proliferation, Migration and Chemotaxis We previously reported that rat microglia are migratory, with most cells having a unipolar morphology with a fan-shaped lamellum at the leading edge and a trailing uropod whether unstimulated or stimulated with IL-4 or IL-10 (Siddiqui et al, 2012, 2014; Vincent et al, 2012; Lively and Schlichter, 2013)

Summary

Introduction

Members of the Kir inward-rectifier K+ channel family (which includes Kir2.1) are expressed in both excitable and nonexcitable cells, where their primary function is to maintain a hyperpolarized membrane potential (Lu, 2004; Hibino et al, 2010). After CNS damage, microglia can enter anti-inflammatory states that help resolve classical activation and promote repair (reviewed in Hanisch and Kettenmann, 2007; Colton, 2009; Czeh et al, 2011). It is not known if Kir2.1 is expressed in microglia in these states; i.e., following ‘‘alternative’’ activation (evoked by interleukin-4) or ‘‘acquired deactivation’’ (evoked by IL10). After CNS injury, the population of microglia at damage sites will depend on both proliferation and migration; it is important to compare these functions in proand anti-inflammatory states. While suggestive of a link between Kir2.1, proliferation and Ca2+ signaling, previous studies have not addressed whether the microglial activation state affects Kir2.1 contributions

Methods

Results

Conclusion