Xie Xinmin, Van Deusen Amy L, Vitko Iuliia, Babu Daniella A, Davies Lucinda A, Huynh Nhung, Cheng Holden, Yang Naibo, Barrett Paula Q, Perez-Reyes Edward
Bioscience Division, SRI International, Menlo Park, CA 94250, USA.
Assay Drug Dev Technol. 2007 Apr;5(2):191-203. doi: 10.1089/adt.2006.054.
T-type Ca(2+) channels encoded by voltage-gated Ca(2+) channel (Ca(v)) 3.1, 3.2, and 3.3 genes play important physiological roles and serve as therapeutic targets for neurological and cardiovascular disorders. Currently there is no selective T-channel blocker. To screen for such a blocker, we developed three stable cell lines expressing human recombinant Ca(v)3.1, 3.2, or 3.3 channels and then examined their usefulness in high throughput screens. All three cell lines displayed an increase in intracellular Ca(2+) in response to changes in extracellular Ca(2+) as detected with Ca(2+)-sensitive dyes using a fluorometric imaging plate reader (FLIPR [Molecular Devices, Sunnyvale, CA] or FlexStation [Molecular Devices]). The signal-to-noise ratio was 2-4. Co-expression of Ca(v)3.2 with a mouse leak K(+) channel, which by virtue of being open at rest hyperpolarizes the cell membrane, blocked the fluorescent signal. Co-addition of KCl to these cells induced a Ca(2+) signal that was similar to that observed in the cell line expressing Ca(v)3.2 alone. These results confirm that the detection of intracellular Ca(2+) increase in cells expressing Ca(v)3.2 alone results from Ca(2+) entry through channels that are open at the resting membrane potential of each cell line (i.e., window currents). Testing known drugs on Ca(v)3 channels showed that block could be reliably detected using the FlexStation assay, FLIPR assay, or voltage clamp recordings using the IonWorks HT system (Molecular Devices). These results support the use of the FLIPR window current assay for primary drug screening and high throughput patch recordings for secondary screening of novel T-channel blockers.
由电压门控钙通道（Ca(v)）3.1、3.2和3.3基因编码的T型钙（Ca(2+)）通道发挥着重要的生理作用，并且是神经和心血管疾病的治疗靶点。目前尚无选择性T通道阻滞剂。为了筛选此类阻滞剂，我们构建了三种稳定表达人重组Ca(v)3.1、3.2或3.3通道的细胞系，然后检测它们在高通量筛选中的效用。使用荧光成像微孔板读数仪（FLIPR [Molecular Devices公司，加利福尼亚州桑尼维尔市] 或FlexStation [Molecular Devices公司]），通过Ca(2+)敏感染料检测发现，所有这三种细胞系在细胞外Ca(2+)变化时细胞内Ca(2+)均增加。信噪比为2至4。Ca(v)3.2与小鼠泄漏钾（K(+)）通道共表达，该钾通道由于在静息时开放使细胞膜超极化，从而阻断了荧光信号。向这些细胞中共同添加氯化钾可诱导产生与单独表达Ca(v)3.2的细胞系中观察到的相似的Ca(2+)信号。这些结果证实，单独表达Ca(v)3.2的细胞中细胞内Ca(2+)增加的检测是由于Ca(2+)通过在每个细胞系静息膜电位时开放的通道进入细胞（即窗电流）所致。在Ca(v)3通道上测试已知药物表明，使用FlexStation检测法、FLIPR检测法或使用IonWorks HT系统（Molecular Devices公司）进行电压钳记录能够可靠地检测到阻滞作用。这些结果支持使用FLIPR窗电流检测法进行初步药物筛选，以及使用高通量膜片钳记录进行新型T通道阻滞剂的二次筛选。
