Small Molecules for Early Endosome-Specific Patch Clamping
Small Molecules for Early Endosome-Specific Patch Clamping
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DOI：
10.1016/j.chembiol.2017.05.025
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发表时间：
2017-07-20
影响因子：
8.6
通讯作者：
Grimm, Christian
中科院分区：
文献类型：
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作者：
Chen, Cheng-Chang;Butz, Elisabeth S.;Grimm, Christian
为了解决内溶酶体离子通道的亚细胞分布问题，我们建立了一种新的实验方法，选择性膜片钳 Rab5 阳性早期内涵体 (EE) 与 Rab7/LAMP1 阳性晚期内涵体/溶酶体 (LE/LY)。为了利用膜片钳技术对内溶酶体膜中的离子通道进行功能表征，开发选择性放大各自细胞器的技术非常重要。我们在这里发现两种小分子，wortmannin 和 latrunculin B，在组合时会扩大 Rab5 阳性 EE，但不会扩大 Rab7-、LAMP1- 或 Rab11 (RE) 阳性囊泡。与遗传方法或以前使用的化合物（例如放大 EE、RE 和 LE/LY 的液泡蛋白）相比，这两种化合物作用迅速、具体，并且易于应用。我们在这里应用这种方法来测量 TRPML 通道介导的电流，特别是 TRPML3，我们发现 TRPML3 在过度表达细胞的 EE 和 LE/LY 以及内源表达 CD11b+ 肺组织巨噬细胞中具有功能活性。
To resolve the subcellular distribution of endolysosomal ion channels, we have established a novel experimental approach to selectively patch clamp Rab5 positive early endosomes (EE) versus Rab7/LAMP1-positive late endosomes/lysosomes (LE/LY). To functionally characterize ion channels in endolysosomal membranes with the patch-clamp technique, it is important to develop techniques to selectively enlarge the respective organelles. We found here that two small molecules, wortmannin and latrunculin B, enlarge Rab5-positive EE when combined but not Rab7-, LAMP1-, or Rab11 (RE)positive vesicles. The two compounds act rapidly, specifically, and are readily applicable in contrast to genetic approaches or previously used compounds such as vacuolin, which enlarges EE, RE, and LE/LY. We apply this approach here to measure currents mediated by TRPML channels, in particular TRPML3, which we found to be functionally active in both EE and LE/LY in overexpressing cells as well as in endogenously expressing CD11b+ lung-tissue macrophages.