Olsen M L, Schade S, Lyons S A, Amaral M D, Sontheimer H
Department of Neurobiology and Civitan International Research Center, University of Alabama at Birmingham, Birmingham, Alabama 35294, USA.
J Neurosci. 2003 Jul 2;23(13):5572-82. doi: 10.1523/JNEUROSCI.23-13-05572.2003.
Voltage-gated chloride channels have recently been implicated as being important for cell proliferation and invasive cell migration of primary brain tumors cells. In the present study we provide several lines of evidence that glioma Cl- currents are primarily mediated by ClC-2 and ClC-3, two genes that belong to the ClC superfamily. Transcripts for ClC-2 thru ClC-7 were detected in a human glioma cell line by PCR, whereas only ClC-2, ClC-3, and ClC-5 protein could be identified by Western blot. Prominent ClC-2, -3, and -5 channel expression was also detected in acute patient biopsies from low- and high-grade malignant gliomas. Immunogold electron microscopic studies as well as digital confocal imaging localized a portion of these ClC channels to the plasma membrane. Whole-cell patch-clamp recordings show the presence of two pharmacologically and biophysically distinct Cl- currents that could be specifically reduced by 48 hr exposure of cells to channel-specific antisense oligonucleotides. ClC-3 antisense selectively and significantly reduced the expression of outwardly rectifying current with pronounced voltage-dependent inactivation. Such currents were sensitive to DIDS (200-500 microm) and 5-nitro-2-(3-phenylpropylamino) benzoic acid (165 microm). ClC-2 antisense significantly reduced expression of inwardly rectifying currents, which were potentiated by hyperpolarizing prepulses and inhibited by Cd2+ (200-500 microm). Currents that were mediated by ClC-5 could not be demonstrated. We suggest that ClC-2 and ClC-3 channels are specifically upregulated in glioma membranes and endow glioma cells with an enhanced ability to transport Cl-. This may in turn facilitate rapid changes in cell size and shape as cells divide or invade through tortuous extracellular brain spaces.
电压门控氯离子通道最近被认为对原发性脑肿瘤细胞的增殖和侵袭性细胞迁移很重要。在本研究中，我们提供了几条证据表明胶质瘤氯离子电流主要由ClC-2和ClC-3介导，这两个基因属于ClC超家族。通过PCR在人胶质瘤细胞系中检测到ClC-2至ClC-7的转录本，而通过蛋白质印迹法仅能鉴定出ClC-2、ClC-3和ClC-5蛋白。在低级别和高级别恶性胶质瘤患者的急性活检样本中也检测到了显著的ClC-2、-3和-5通道表达。免疫金电子显微镜研究以及数字共聚焦成像将这些ClC通道的一部分定位到质膜上。全细胞膜片钳记录显示存在两种药理学和生物物理学上不同的氯离子电流，细胞暴露于通道特异性反义寡核苷酸48小时后，这些电流可被特异性降低。ClC-3反义寡核苷酸选择性且显著降低了具有明显电压依赖性失活的外向整流电流的表达。这种电流对DIDS（200 - 500微米）和5-硝基-2-(3-苯基丙基氨基)苯甲酸（165微米）敏感。ClC-2反义寡核苷酸显著降低了内向整流电流的表达，内向整流电流被超极化预脉冲增强并被Cd2+（200 - 500微米）抑制。无法证明存在由ClC-5介导的电流。我们认为ClC-2和ClC-3通道在胶质瘤膜中特异性上调，赋予胶质瘤细胞增强的氯离子转运能力。这反过来可能有助于细胞在分裂或通过曲折的细胞外脑间隙侵袭时，细胞大小和形状的快速变化。
