Department of Neurobiology and Center for Glial Biology in Medicine, University of Alabama at Birmingham, Birmingham, Alabama 35294, USA.
J Biol Chem. 2010 Apr 9;285(15):11188-96. doi: 10.1074/jbc.M109.097675. Epub 2010 Feb 5.
Glioblastoma multiforme is the most common and lethal primary brain cancer in adults. Tumor cells diffusely infiltrate the brain making focal surgical and radiation treatment challenging. The invasion of glioma cells into normal brain is facilitated by the activity of ion channels aiding dynamic regulation of cell volume. Recent studies have specifically implicated ClC-3, a voltage-gated chloride channel, in this process. However, the interaction between ClC-3 activity and cell movement is poorly understood. Here, we demonstrate that ClC-3 is highly expressed on the plasma membrane of human glioma cells where its activity is regulated through phosphorylation via Ca(2+)/calmodulin-dependent protein kinase II (CaMKII). Intracellular infusion of autoactivated CaMKII via patch pipette enhanced chloride currents 3-fold, and this regulation was inhibited by autocamtide-2 related inhibitory peptide, a CaMKII-specific inhibitor. CaMKII modulation of chloride currents was also lost upon stable small hairpin RNA knockdown of ClC-3 channels indicating a specific interaction of ClC-3 and CaMKII. In ClC-3-expressing cells, inhibition of CaMKII reduced glioma invasion to the same extent as direct inhibition of ClC-3. The importance of the molecular interaction of ClC-3 and CaMKII is further supported by our finding that CaMKII co-localizes and co-immunoprecipitates with ClC-3. ClC-3 and CaMKII also co-immunoprecipitate in tissue biopsies from patients diagnosed with grade IV glioblastoma. These tumor samples show 10-fold higher ClC-3 protein expression than nonmalignant brain. These data suggest that CaMKII is a molecular link translating intracellular calcium changes, which are intrinsically associated with glioma migration, to changes in ClC-3 conductance required for cell movement.
多形性胶质母细胞瘤是成人中最常见和最致命的原发性脑癌。肿瘤细胞弥漫性浸润大脑，使局部手术和放射治疗具有挑战性。离子通道的活性有助于细胞体积的动态调节，从而促进神经胶质瘤细胞侵入正常大脑。最近的研究特别表明，电压门控氯离子通道 ClC-3 参与了这一过程。然而，ClC-3 活性与细胞运动之间的相互作用还知之甚少。在这里，我们证明 ClC-3 在人神经胶质瘤细胞的质膜上高度表达，其活性通过 Ca（2+）/钙调蛋白依赖性蛋白激酶 II（CaMKII）磷酸化调节。通过膜片钳内灌流自动激活的 CaMKII 通过贴壁管增强氯离子电流 3 倍，这种调节被 CaMKII 特异性抑制剂 autocamtide-2 相关抑制肽抑制。ClC-3 通道的稳定短发夹 RNA 敲低也丧失了 CaMKII 对氯离子电流的调节，表明 ClC-3 和 CaMKII 之间存在特异性相互作用。在 ClC-3 表达细胞中，CaMKII 抑制抑制神经胶质瘤侵袭的程度与直接抑制 ClC-3 相同。ClC-3 和 CaMKII 之间的分子相互作用的重要性还得到了我们的发现的支持，即 CaMKII 与 ClC-3 共定位并共免疫沉淀。CaMKII 和 ClC-3 也在诊断为 IV 级胶质母细胞瘤的患者组织活检中共同免疫沉淀。这些肿瘤样本的 ClC-3 蛋白表达比非恶性脑组织高 10 倍。这些数据表明，CaMKII 是一种分子连接，它将内在与神经胶质瘤迁移相关的细胞内钙变化转化为细胞运动所需的 ClC-3 电导变化。
