A radioactive uptake assay to measure ion transport across ion channel-containing liposomes
A radioactive uptake assay to measure ion transport across ion channel-containing liposomes
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DOI：
10.1038/nprot.2006.166
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发表时间：
2006-01-01
期刊：
影响因子：
14.8
通讯作者：
Nimigean, Crina M.
中科院分区：
文献类型：
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作者：
Nimigean, Crina M.
在这里，我们描述了一种将离子通道整合到脂泡(脂质体)中的程序，并通过分析这些蛋白脂质体中的放射性同位素摄取来确定通道种群的功能。所描述的技术将仅适用于钾通道，但可以容易地修改，如正文中所建议的，用于其他离子通道和转运体。洗涤剂胶束中的纯化离子通道蛋白与溶解的脂类结合。洗涤剂随后通过凝胶过滤或渗析到高钾(高[K+])缓冲液中从蛋白质-脂复合体中去除。在冻融和超声化后，生成的较大的脂质体通过另一个凝胶过滤柱，将脂外高的[K+]交换到低的[K+]缓冲液，从而在脂质体的膜上建立大的K+梯度。然后将微量RB-86加入到脂外空间中，反应开始。如果离子通道对K+具有通透性，则内部的K+沿着其浓度梯度退出脂质体，而外部的Rb-86则聚集在脂质体内空间，直到达到平衡。通过测量在离子交换柱上除去外部RB-86后累积的RB-86来监测反应时间过程。RB-86助熔剂分析需要2-5个小时，具体取决于反应速度和所需时间点的数量。
Here we describe a procedure for incorporating ion channels into lipid vesicles (liposomes) and functional characterization of the channel population by assaying radioactive isotope uptake into these proteoliposomes. The technique as described will work only for potassium channels but can be easily modified, as suggested in the text, for other ion channels and transporters. Purified ion channel proteins in detergent micelles are combined with solubilized lipids. Detergent is subsequently removed from protein-lipid complexes by gel filtration or dialysis into high potassium (high [K+]) buffer. After freezing-thawing and sonication, the resultant larger liposomes are passed over another gel-filtration column to exchange an extraliposomal high [K+] to a low [K+] buffer, thus establishing a large K+ gradient across the liposomal membrane. Trace Rb-86 is then added to the extraliposomal space and the reaction begins. If the ion channel is permeable to K+, the K+ inside exits the liposomes down its concentration gradient and the Rb-86 outside accumulates in the intraliposomal space until equilibrium is reached. The reaction time course is monitored by measurement of accumulated Rb-86 after removal of external Rb-86 over an ion-exchange column. The Rb-86 flux assay takes 2-5 hours depending on the reaction rate and the number of desired time points.