A novel fluorescent toxin to detect and investigate Kv1.3 channel up-regulation in chronically activated T lymphocytes
A novel fluorescent toxin to detect and investigate Kv1.3 channel up-regulation in chronically activated T lymphocytes
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DOI：
10.1074/jbc.m212868200
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发表时间：
2003-03-14
影响因子：
4.8
通讯作者：
Chandy, KG
中科院分区：
文献类型：
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作者：
Beeton, C;Wulff, H;Chandy, KG
电压门控Kv1.3通道异常高表达的T淋巴细胞（Kv1.3高细胞）与实验性自身免疫性脑脊髓炎（多发性硬化症的动物模型）的发病机制有关。我们开发了一种荧光类似物ShK (ShK- f6ca)，它是已知最有效的Kv1.3抑制剂，用于流式细胞术检测Kv1.3高细胞。ShK-F6CA在皮摩尔浓度下阻断Kv1.3，希尔系数为1，对Kv1.3的特异性是Kv1.1和其他K-v通道的80倍。在流式细胞术实验中，ShK-F6CA特异性染色表达kv1.3的细胞，检测限接近每个细胞600个通道。经抗原反复刺激7-10次的大鼠和人T细胞与静息T细胞或经过1-3轮活化的细胞相比，Kv1.3通道（bbb600通道/细胞）和ShK-F6CA染色水平较高，很容易区分。髓鞘抗原刺激后，功能性Kv1.3表达水平在髓鞘特异性大鼠T细胞系中显著增加，在15-20小时达到峰值，然后在接下来的7天内下降到基线水平，与脑致原性的获得和丧失同时发生。抗原诱导的Kv1.3上调需要钙和蛋白激酶c依赖途径。ShK-F6CA可用于正常和病变组织中Kv1.3（高）表达细胞的快速定量检测，以及完整细胞中功能通道的可视化分布。
T lymphocytes with unusually high expression of the voltage-gated Kv1.3 channel (Kv1.3 high cells) have been implicated in the pathogenesis of experimental autoimmune encephalomyelitis, an animal model for multiple sclerosis. We have developed a fluoresceinated analog of ShK (ShK-F6CA), the most potent known inhibitor of Kv1.3, for detection of Kv1.3 high cells by flow cytometry. ShK-F6CA blocked Kv1.3 at picomolar concentrations with a Hill coefficient of I and exhibited >80-fold specificity for Kv1.3 over Kv1.1 and other K-v channels. In flow cytometry experiments, ShK-F6CA specifically stained Kv1.3-expressing cells with a detection limit of similar to600 channels per cell. Rat and human T cells that had been repeatedly stimulated 7-10 times with antigen were readily distinguished on the basis of their high levels of Kv1.3 channels (>600 channels/cell) and ShK-F6CA staining from resting T cells or cells that had undergone 1-3 rounds of activation. Functional Kv1.3 expression levels increased substantially in a myelinspecific rat T cell line following myelin antigen stimulation, peaking at 15-20 h and then declining to baseline over the next 7 days, in parallel with the acquisition and loss of encephalitogenicity. Both calcium- and protein kinase C-dependent pathways were required for the antigen-induced Kv1.3 up-regulation. ShK-F6CA might be useful for rapid and quantitative detection of Kv1.3(high) expressing cells in normal and diseased tissues, and to visualize the distribution of functional channels in intact cells.