Patch-clamp technique to characterize ion channels in enlarged individual endolysosomes
Patch-clamp technique to characterize ion channels in enlarged individual endolysosomes
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DOI：
10.1038/nprot.2017.036
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发表时间：
2017-08-01
期刊：
影响因子：
14.8
通讯作者：
Grimm, Christian
中科院分区：
文献类型：
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作者：
Chen, Cheng-Chang;Cang, Chunlei;Grimm, Christian
根据蛋白质组学分析，超过70种不同的离子通道和转运蛋白存在于细胞内区室的膜中，例如内体和溶酶体。这些通道的功能障碍与人类疾病如溶酶体贮积症、神经退行性疾病和代谢病理学以及某些传染病的进展有关。因此，这些通道作为未来的药物靶点引起了非常高的兴趣。缺乏细胞内离子通道的详细电生理表征，主要是因为分析质膜离子通道的标准方法，如膜片钳技术，不容易适用于细胞内细胞器。在这里，我们提出了一个协议，详细介绍了如何实现手动膜片钳技术的内溶酶体室。与交替使用的平面内溶酶体膜片钳技术相比，该方法是一种视觉控制的直接膜片钳技术，类似于传统的膜片钳。该方案假设具有膜片钳方法的基本知识和经验。该方法的实施需要长达1周，材料准备需要2-4天。一个单独的实验（即，测量穿过内溶酶体膜的通道电流），包括对照实验，可以在1小时内完成。这不包括内溶酶体扩大的时间，其需要1至48小时，取决于所使用的方法和细胞类型。数据分析需要一个小时。
According to proteomics analyses, more than 70 different ion channels and transporters are harbored in membranes of intracellular compartments such as endosomes and lysosomes. Malfunctioning of these channels has been implicated in human diseases such as lysosomal storage disorders, neurodegenerative diseases and metabolic pathologies, as well as in the progression of certain infectious diseases. As a consequence, these channels have engendered very high interest as future drug targets. Detailed electrophysiological characterization of intracellular ion channels is lacking, mainly because standard methods to analyze plasma membrane ion channels, such as the patch-clamp technique, are not readily applicable to intracellular organelles. Here we present a protocol detailing how to implement a manual patch-clamp technique for endolysosomal compartments. In contrast to the alternatively used planar endolysosomal patch-clamp technique, this method is a visually controlled, direct patch-clamp technique similar to conventional patch-clamping. The protocol assumes basic knowledge and experience with patch-clamp methods. Implementation of the method requires up to 1 week, and material preparation takes similar to 2-4 d. An individual experiment (i.e., measurement of channel currents across the endolysosomal membrane), including control experiments, can be completed within 1 h. This excludes the time for endolysosome enlargement, which takes between 1 and 48 h, depending on the approach and cell type used. Data analysis requires an additional hour.
DOI：
10.1083/jcb.201610067
发表时间：
2016-11-07
期刊：
The Journal of cell biology
影响因子：
--
作者：
Gu M;Xu H
通讯作者：
Xu H