Baxter Deborah F, Kirk Martin, Garcia Amy F, Raimondi Alejandra, Holmqvist Mats H, Flint Kimberly K, Bojanic Dejan, Distefano Peter S, Curtis Rory, Xie Yu
Millennium Pharmaceuticals, Inc., Cambridge, MA 02139, USA.
J Biomol Screen. 2002 Feb;7(1):79-85. doi: 10.1177/108705710200700110.
The study of ion channel-mediated changes in membrane potential using the conventional bisoxonol fluorescent dye DiBAC(4)(3) has several limitations, including a slow onset of response and multistep preparation, that limit both the fidelity of the results and the throughput of membrane potential assays. Here, we report the characterization of the FLIPR Membrane Potential Assay Kit (FMP) in cells expressing voltage- and ligand-gated ion channels. The steady-state and kinetics fluorescence properties of FMP were compared with those of DiBAC(4)(3), using both FLIPR and whole-cell patch-clamp recording. Our experiments with the voltage-gated K(+) channel, hElk-1, revealed that FMP was 14-fold faster than DiBAC(4)(3) in response to depolarization. On addition of 60 mM KCl, the kinetics of fluorescence changes of FMP using FLIPR were identical to those observed in the electrophysiological studies using whole-cell current clamp. In addition, KCl concentration-dependent increases in FMP fluorescence correlated with the changes of membrane potential recorded in whole-cell patch clamp. In studies examining vanilloid receptor-1, a ligand-gated nonselective cation channel, FMP was superior to DiBAC(4)(3) with respect to both kinetics and amplitude of capsaicin-induced fluorescence changes. FMP has also been used to measure the activation of K(ATP) and hERG. Thus this novel membrane potential dye represents a powerful tool for developing high-throughput screening assays for ion channels.
使用传统的双羟萘酚荧光染料DiBAC(4)(3)研究离子通道介导的膜电位变化存在若干局限性，包括反应起始缓慢和多步骤制备过程，这些局限性既限制了结果的准确性，也限制了膜电位检测的通量。在此，我们报告了在表达电压门控和配体门控离子通道的细胞中对荧光成像仪膜电位检测试剂盒（FMP）的特性描述。使用荧光成像仪和全细胞膜片钳记录，将FMP的稳态和动力学荧光特性与DiBAC(4)(3)的进行了比较。我们对电压门控钾通道hElk-1的实验表明，在去极化反应中，FMP比DiBAC(4)(3)快14倍。加入60 mM KCl后，使用荧光成像仪检测FMP荧光变化的动力学与使用全细胞电流钳进行电生理研究中观察到的一致。此外，FMP荧光随KCl浓度的增加与全细胞膜片钳记录的膜电位变化相关。在研究香草酸受体-1（一种配体门控非选择性阳离子通道）时，FMP在辣椒素诱导的荧光变化的动力学和幅度方面均优于DiBAC(4)(3)。FMP还被用于测量K(ATP)和hERG的激活。因此，这种新型膜电位染料是开发离子通道高通量筛选检测方法的有力工具。
