A novel membrane potential-sensitive fluorescent dye improves cell-based assays for ion channels
A novel membrane potential-sensitive fluorescent dye improves cell-based assays for ion channels
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DOI：
10.1177/108705710200700110
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发表时间：
2002-02-01
影响因子：
--
通讯作者：
Xie, Y
中科院分区：
文献类型：
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作者：
Baxter, DF;Kirk, M;Xie, Y
使用传统的双酚A荧光染料DiBAC（4）（3）研究离子通道介导的膜电位变化有几个局限性，包括反应开始缓慢和多步制备，这限制了结果的保真度和膜电位测定的通量。在这里，我们报告的FLIPR膜电位测定试剂盒（FMP）在细胞表达电压和配体门控离子通道的表征。使用FLIPR和全细胞膜片钳记录，比较了FMP与DiBAC的稳态和动力学荧光特性（4）（3）。我们对电压门控K+通道hElk-1的实验显示，FMP对去极化的反应比DiBAC快14倍（4）（3）。在添加60 mM KCl时，使用FLIPR的FMP荧光变化的动力学与使用全细胞电流钳的电生理研究中观察到的那些相同。此外，KCl浓度依赖性增加FMP荧光与全细胞膜片钳记录的膜电位的变化。在研究香草素受体-1（一种配体门控非选择性阳离子通道）的研究中，FMP在辣椒素诱导的荧光变化的动力学和幅度方面均上级DiBAC 4（3）。FMP还用于测量K-ATP（1）和hERG的激活。(2)因此，这种新型膜电位染料代表了开发离子通道高通量筛选分析的强大工具。
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 DiBAC4(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(1) and hERG.(2) Thus this novel membrane potential dye represents a powerful tool for developing high-throughput screening assays for ion channels.