Department of Pharmacy, Center for Drug Research and Center for Integrated Protein Science Munich (CIPSM), Ludwig-Maximilians-Universität München, 81377 Munich, Germany.
Department of Neurology, Center for Human Genetic Research, Massachusetts General Hospital, Harvard University, Boston, MA 02114, USA.
Cell Chem Biol. 2017 Jul 20;24(7):907-916.e4. doi: 10.1016/j.chembiol.2017.05.025.
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.
为了解析内体溶酶体离子通道的亚细胞分布，我们建立了一种新的实验方法，用于选择性地对 Rab5 阳性早期内体 (EE) 与 Rab7/LAMP1 阳性晚期内体/溶酶体 (LE/LY) 进行膜片钳封接。为了用膜片钳技术对溶酶体膜中的离子通道进行功能表征，开发选择性扩大相应细胞器的技术非常重要。我们在这里发现，两种小分子化合物wortmannin 和 latrunculin B 联合使用时可扩大 Rab5 阳性 EE，但不能扩大 Rab7、LAMP1 或 Rab11（RE）阳性囊泡。这两种化合物作用迅速、特异性强，与遗传方法或以前使用的化合物（如 vacuolin，可扩大 EE、RE 和 LE/LY）相比，更容易应用。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，我们发现它在过表达细胞中的 EE 和 LE/LY 中以及内源性表达 CD11b+肺组织巨噬细胞中均具有功能活性。
