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.

中文翻译：

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小分子，用于早期内体特异性膜片钳

为了解决溶酶体离子通道的亚细胞分布，我们建立了一种新颖的实验方法来选择性地修补钳制Rab5阳性早期内体（EE）与Rab7 / LAMP1阳性晚期内体/溶酶体（LE / LY）。为了用膜片钳技术在功能上表征溶酶体膜中的离子通道，重要的是开发能够选择性扩大各个细胞器的技术。我们在这里发现，两个小分子渥曼青霉素和拉特古林B结合时会增加Rab5阳性EE，但不会增加Rab7-，LAMP1-或Rab11（RE）阳性囊泡。相对于遗传方法或先前使用的化合物（例如，增加EE，RE和LE / LY的液泡蛋白），这两种化合物的作用特别迅速，并且易于应用。我们在这里采用这种方法来测量TRPML通道介导的电流，