Terstappen G C
Bayer AG, Pharma Research Centre, Wuppertal, D-42096, Germany.
Anal Biochem. 1999 Aug 1;272(2):149-55. doi: 10.1006/abio.1999.4179.
A nonradioactive cell-based rubidium (Rb(+)) efflux assay for functional analysis of native and recombinant ion channels has been developed. Cells are first loaded with rubidium, a tracer for potassium, and after channel activation, rubidium distribution between intracellular and extracellular space is determined by atomic absorption spectroscopy. The relative amount of rubidium in the cell supernatant is a direct measure of channel activity. The broad utility of the method is demonstrated by analysis of a range of different ion channels. Ligand-gated ion channels like nicotinic acetylcholine receptors and purinergic P2X receptors were studied in native PC-12 cells. Calcium-activated potassium channels were analyzed in native (small-conductance calcium-activated potassium channel, SK(Ca)) as well as recombinant cell lines (large-conductance calcium-activated potassium channel, BK(Ca)). Also recombinant voltage-gated potassium channels (Kv1.1, Kv1.4) were amenable to this functional analysis. The method is particularly useful for identification of ion channel modulators in drug discovery since it allows functional analysis with high capacity.
已开发出一种基于非放射性细胞的铷（Rb(+)）外流测定法，用于天然和重组离子通道的功能分析。首先将细胞用铷（钾的一种示踪剂）加载，在通道激活后，通过原子吸收光谱法测定细胞内和细胞外空间之间的铷分布。细胞上清液中铷的相对量是通道活性的直接度量。通过对一系列不同离子通道的分析证明了该方法的广泛实用性。在天然PC-12细胞中研究了配体门控离子通道，如烟碱型乙酰胆碱受体和嘌呤能P2X受体。在天然细胞（小电导钙激活钾通道，SK(Ca)）以及重组细胞系（大电导钙激活钾通道，BK(Ca)）中分析了钙激活钾通道。同样，重组电压门控钾通道（Kv1.1、Kv1.4）也适用于这种功能分析。该方法对于药物发现中离子通道调节剂的鉴定特别有用，因为它允许进行高通量功能分析。
