Small Molecules for Early Endosome-Specific Patch Clamping. Issue 7 (20th July 2017)

• Record Type: Journal Article
• Authors: Chen, Cheng-Chang; Butz, Elisabeth S.; Chao, Yu-Kai; Grishchuk, Yulia; Becker, Lars; Heller, Stefan; Slaugenhaupt, Susan A.; Biel, Martin; Wahl-Schott, Christian; Grimm, Christian
• Is Part Of: Cell chemical biology. Volume 24:Issue 7(2017)
• Page Start: 907
• Page End: 916.e4
• Publication Date: 2017-07-20
• Subjects: endosome -- TRPML1 -- TRPML3 -- TRPML -- latrunculin B -- wortmannin; Biochemistry -- Periodicals; 572.05
• DOI: 10.1016/j.chembiol.2017.05.025
• Languages: English
• ISSNs: 2451-9456

Abstract

Summary: 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. Graphical Abstract: Highlights: A novel approach for early-endosome-selective patch clamping is provided Wortmannin and latrunculin B enable selective EE patch-clamp experimentation TRPML channels are characterized in EE and LE/LY TRPML3 is shown to be functionally active in both EE and LE/LY Abstract : To functionally characterize ion channels in endolysosomal membranes with the patch-clamp technique, it is important to develop techniques to selectively enlarge organelles. Chen et al. found here that wortmannin and latrunculin B specifically enlarge Rab5-positive early endosomes.