Quantification of the functional expression of the Ca2+-activated K+channel KCa3.1 on microglia from adult human neocortical tissue

Blomster, Linda V.; Strøbæk, Dorte; Hougaard, Charlotte; Klein, Jessica; Pinborg, Lars H.; Mikkelsen, Jens D.; Christophersen, Palle

doi:10.1002/glia.23040

Cited by 20 publications

(9 citation statements)

References 40 publications

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“…While human T cells already express roughly 250 K V 1.3 channels in the resting state, rat and mouse T cells typically only express a very small number of channels (∼5) in the resting state and then up‐regulate K V 1.3 expression after activation (Beeton and Chandy, 2005; Beeton et al, ; Decoursey et al, ). Interestingly, another group very recently patch‐clamped adult human microglia from neocortical tissue surgically removed from epilepsy patients and found high K Ca 3.1 current densities (∼580 per cell), which, similar to our observations here, did not significantly change with LPS or IL‐4 treatment (Blomster et al, ).…”

Section: Discussionsupporting

confidence: 90%

Differential Kv1.3, KCa3.1, and Kir2.1 expression in “classically” and “alternatively” activated microglia

Nguyen

Grössinger

Horiuchi

et al. 2016

Glia

131

121

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Microglia are highly plastic cells that can assume different phenotypes in response to microenvironmental signals. Lipopolysaccharide (LPS) and interferon‐γ (IFN‐γ) promote differentiation into classically activated M1‐like microglia, which produce high levels of pro‐inflammatory cytokines and nitric oxide and are thought to contribute to neurological damage in ischemic stroke and Alzheimer's disease. IL‐4 in contrast induces a phenotype associated with anti‐inflammatory effects and tissue repair. We here investigated whether these microglia subsets vary in their K+ channel expression by differentiating neonatal mouse microglia into M(LPS) and M(IL‐4) microglia and studying their K+ channel expression by whole‐cell patch‐clamp, quantitative PCR and immunohistochemistry. We identified three major types of K+ channels based on their biophysical and pharmacological fingerprints: a use‐dependent, outwardly rectifying current sensitive to the KV1.3 blockers PAP‐1 and ShK‐186, an inwardly rectifying Ba2+‐sensitive Kir2.1 current, and a Ca2+‐activated, TRAM‐34‐sensitive KCa3.1 current. Both KV1.3 and KCa3.1 blockers inhibited pro‐inflammatory cytokine production and iNOS and COX2 expression demonstrating that KV1.3 and KCa3.1 play important roles in microglia activation. Following differentiation with LPS or a combination of LPS and IFN‐γ microglia exhibited high KV1.3 current densities (∼50 pA/pF at 40 mV) and virtually no KCa3.1 and Kir currents, while microglia differentiated with IL‐4 exhibited large Kir2.1 currents (∼ 10 pA/pF at −120 mV). KCa3.1 currents were generally low but moderately increased following stimulation with IFN‐γ or ATP (∼10 pS/pF). This differential K+ channel expression pattern suggests that KV1.3 and KCa3.1 inhibitors could be used to inhibit detrimental neuroinflammatory microglia functions. GLIA 2016;65:106–121

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Section: Discussionsupporting

confidence: 90%

Differential Kv1.3, KCa3.1, and Kir2.1 expression in “classically” and “alternatively” activated microglia

Nguyen

Grössinger

Horiuchi

et al. 2016

Glia

131

121

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“…A recent article reported that neocortical microglia isolated and cultured from human epilepsy patients responded to LPS with an increase in TNF-α production but no change in KCa3.1 expression. In contrast, IL-4 slightly increased the KCa3.1 current per cell, although no significant change in channel density was observed (Blomster et al 2016). Thus, although previous studies using cultured neonatal microglia suggest a pro-inflammatory role of KCa3.1 (Kaushal et al 2007; Maezawa et al 2011; Schlichter et al 2010), its actual functional roles in adults appear complicated and need to be verified by in vivo experiments as exemplified by this study.…”

Section: Discussionmentioning

confidence: 99%

The voltage‐gated potassium channel Kv1.3 is required for microglial pro‐inflammatory activation in vivo

Lucente

Nguyen

Wulff

et al. 2018

Glia

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Microglia show a rich repertoire of activation patterns regulated by a complex ensemble of surface ion channels, receptors, and transporters. We and others have investigated whether microglia vary their K channel expression as a means to achieve functional diversity. However, most of the prior studies were conducted using in vitro models such as BV2 cells, primary microglia, or brain slices in culture, which may not accurately reflect microglia physiology in adult individuals. Here we employed an in vivo mouse model of selective innate immune activation by intracerebroventricular injection of lipopolysaccharides (ICV-LPS) to determine the role of the voltage-gated Kv1.3 channel in LPS-induced M1-like microglial activation. Using microglia acutely isolated from adult brains, we detected Kv1.3 and Kir2.1 currents, and found that ICV-LPS increased the current density and RNA expression of Kv1.3 but did not affect those of Kir2.1. Genetic knockout of Kv1.3 abolished LPS-induced microglial activation exemplified by Iba-1 immunoreactivity and expression of pro-inflammatory mediators such as IL-1β, TNF-α, IL-6, and iNOS. Moreover, Kv1.3 knockout mitigated the LPS-induced impairment of hippocampal long-term potentiation (hLTP), suggesting that Kv1.3 activity regulates pro-inflammatory microglial neurotoxicity. Pharmacological intervention using PAP-1, a small molecule that selectively blocks homotetrameric Kv1.3 channels, achieved anti-inflammatory and hLTP-recovery effects similar to Kv1.3 knockout. We conclude that Kv1.3 is required for microglial M1-like pro-inflammatory activation in vivo. A significant implication of our in vivo data is that Kv1.3 blockers could be therapeutic candidates for neurological diseases where microglia-mediated neurotoxicity is implicated in the pathogenesis.

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“…For instance, no KCa3.1 current was seen in juvenile mouse microglia within acute brain slices (Schilling and Eder, 2007 ); whereas in adult microglia isolated using CD11b-coated magnetic beads, there were KCa3.1 currents in some microglia at 8 days after ischemia, and in most microglia isolated from LPS-injected mouse brains (Chen et al, 2016 ). In adult human microglia from epilepsy patients, most cells expressed a KCa3.1 current, which was unaffected by LPS and slightly increased by IL-4 (Blomster et al, 2016 ). Thus, a clear picture of KCa3.1 expression in microglia has not emerged.…”

Section: Discussionmentioning

confidence: 99%

Comparing Effects of Transforming Growth Factor β1 on Microglia From Rat and Mouse: Transcriptional Profiles and Potassium Channels

Lively

Lam

Wong

et al. 2018

Front. Cell. Neurosci.

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The cytokine, transforming growth factor β1 (TGFβ1), is up-regulated after central nervous system (CNS) injuries or diseases involving microglial activation, and it has been proposed as a therapeutic agent for treating neuroinflammation. Microglia can produce and respond to TGFβ1. While rats and mice are commonly used for studying neuroinflammation, very few reports directly compare them. Such studies are important for improving pre-clinical studies and furthering translational progress in developing therapeutic interventions. After intracerebral hemorrhage (ICH) in the rat striatum, the TGFβ1 receptor was highly expressed on microglia/macrophages within the hematoma. We recently found species similarities and differences in response to either a pro-inflammatory (interferon-γ, IFN-γ, +tumor necrosis factor, TNF-α) or anti-inflammatory interleukin-4 (IL-4) stimulus. Here, we assessed whether rat and mouse microglia differ in their responses to TGFβ1. Microglia were isolated from Sprague-Dawley rats and C57BL/6 mice and treated with TGFβ1. We quantified changes in expression of >50 genes, in their morphology, proliferation, apoptosis and in three potassium channels that are considered therapeutic targets. Many inflammatory mediators, immune receptors and modulators showed species similarities, but notable differences included that, for some genes, only one species responded (e.g., Il4r, Il10, Tgfbr2, colony-stimulating factor receptor (Csf1r), Itgam, suppressor of cytokine signaling 1 (Socs1), toll-like receptors 4 (Tlr4), P2rx7, P2ry12), and opposite responses were seen for others (Tgfb1, Myc, Ifngr1). In rat only, TGFβ1 affected microglial morphology and proliferation, but there was no apoptosis in either species. In both species, TGFβ1 dramatically increased Kv1.3 channel expression and current (no effects on Kir2.1). KCa3.1 showed opposite species responses: the current was low in unstimulated rat microglia and greatly increased by TGFβ1 but higher in control mouse cells and decreased by TGFβ1. Finally, we compared TGFβ1 and IL10 (often considered similar anti-inflammatory stimuli) and found many different responses in both species. Overall, the numerous species differences should be considered when characterizing neuroinflammation and microglial activation in vitro and in vivo, and when targeting potassium channels.

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Quantification of the functional expression of the Ca²⁺-activated K⁺channel K_Ca3.1 on microglia from adult human neocortical tissue

Cited by 20 publications

References 40 publications

Differential Kv1.3, KCa3.1, and Kir2.1 expression in “classically” and “alternatively” activated microglia

Differential Kv1.3, KCa3.1, and Kir2.1 expression in “classically” and “alternatively” activated microglia

The voltage‐gated potassium channel Kv1.3 is required for microglial pro‐inflammatory activation in vivo

Comparing Effects of Transforming Growth Factor β1 on Microglia From Rat and Mouse: Transcriptional Profiles and Potassium Channels

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