Institute of Pharmacology and Clinical Pharmacy, Philipps University of Marburg, 35032, Marburg, Germany.
Glia. 2012 Dec;60(12):2050-64. doi: 10.1002/glia.22419. Epub 2012 Sep 21.
In neurons, small-conductance calcium-activated potassium (KCNN/SK/K(Ca)2) channels maintain calcium homeostasis after N-methyl-D-aspartate (NMDA) receptor activation, thereby preventing excitotoxic neuronal death. So far, little is known about the function of KCNN/SK/K(Ca)2 channels in non-neuronal cells, such as microglial cells. In this study, we addressed the question whether KCNN/SK/K(Ca)2 channels activation affected inflammatory responses of primary mouse microglial cells upon lipopolysaccharide (LPS) stimulation. We found that N-cyclohexyl-N-[2-(3,5-dimethyl-pyrazol-1-yl)-6-methyl-4-pyrimidinamine (CyPPA), a positive pharmacological activator of KCNN/SK/K(Ca)2 channels, significantly reduced LPS-stimulated activation of microglia in a concentration-dependent manner. The general KCNN/SK/K(Ca)2 channel blocker apamin reverted these effects of CyPPA on microglial proliferation. Since calcium plays a central role in microglial activation, we further addressed whether KCNN/SK/K(Ca)2 channel activation affected the changes of intracellular calcium levels, Ca(2+), in microglial cells. Our data show that LPS-induced elevation of Ca(2+) was attenuated following activation of KCNN2/3/K(Ca)2.2/K(Ca)2.3 channels by CyPPA. Furthermore, CyPPA reduced downstream events including tumor necrosis factor alpha and interleukin 6 cytokine production and nitric oxide release in activated microglia. Further, we applied specific peptide inhibitors of the KCNN/SK/K(Ca)2 channel subtypes to identify which particular channel subtype mediated the observed anti-inflammatory effects. Only inhibitory peptides targeting KCNN3/SK3/K(Ca)2.3 channels, but not KCNN2/SK2/K(Ca)2.2 channel inhibition, reversed the CyPPA-effects on LPS-induced microglial proliferation. These findings revealed that KCNN3/SK3/K(Ca)2.3 channels can modulate the LPS-induced inflammatory responses in microglial cells. Thus, KCNN3/SK3/K(Ca)2.3 channels may serve as a therapeutic target for reducing microglial activity and related inflammatory responses in the central nervous system.
在神经元中，小电导钙激活钾 (KCNN/SK/K(Ca)2) 通道在 N-甲基-D-天冬氨酸 (NMDA) 受体激活后维持钙稳态，从而防止兴奋性神经元死亡。到目前为止，对于 KCNN/SK/K(Ca)2 通道在非神经元细胞（如小胶质细胞）中的功能知之甚少。在这项研究中，我们研究了 KCNN/SK/K(Ca)2 通道的激活是否会影响脂多糖 (LPS) 刺激后原代小鼠小胶质细胞的炎症反应。我们发现，N-环己基-N-[2-(3,5-二甲基-吡唑-1-基)-6-甲基-4-嘧啶胺 (CyPPA)，一种 KCNN/SK/K(Ca)2 通道的阳性药理学激活剂，以浓度依赖的方式显著降低 LPS 刺激的小胶质细胞激活。一般的 KCNN/SK/K(Ca)2 通道阻滞剂 apamin 逆转了 CyPPA 对小胶质细胞增殖的这些影响。由于钙在小胶质细胞激活中起着核心作用，我们进一步研究了 KCNN/SK/K(Ca)2 通道的激活是否会影响小胶质细胞内钙水平 Ca(2+) 的变化。我们的数据表明，CyPPA 激活 KCNN2/3/K(Ca)2.2/K(Ca)2.3 通道后，LPS 诱导的 Ca(2+) 升高减弱。此外，CyPPA 减少了激活的小胶质细胞中肿瘤坏死因子 alpha 和白细胞介素 6 细胞因子产生和一氧化氮释放等下游事件。此外，我们应用了 KCNN/SK/K(Ca)2 通道亚型的特异性肽抑制剂，以确定观察到的抗炎作用是由哪种特定的通道亚型介导的。只有针对 KCNN3/SK3/K(Ca)2.3 通道的抑制性肽，而不是 KCNN2/SK2/K(Ca)2.2 通道的抑制，逆转了 CyPPA 对 LPS 诱导的小胶质细胞增殖的影响。这些发现表明，KCNN3/SK3/K(Ca)2.3 通道可以调节小胶质细胞中 LPS 诱导的炎症反应。因此，KCNN3/SK3/K(Ca)2.3 通道可能成为减少中枢神经系统中小胶质细胞活性和相关炎症反应的治疗靶点。
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