1. Small Molecules for Early Endosome-Specific Patch Clamping.
- Author
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Chen CC, Butz ES, Chao YK, Grishchuk Y, Becker L, Heller S, Slaugenhaupt SA, Biel M, Wahl-Schott C, and Grimm C
- Subjects
- Aminopyridines pharmacology, CD11b Antigen metabolism, Endosomes drug effects, HEK293 Cells, Heterocyclic Compounds, 3-Ring pharmacology, Humans, Lung cytology, Lung metabolism, Lysosomal-Associated Membrane Protein 1 metabolism, Lysosomes drug effects, Lysosomes metabolism, Macrophages cytology, Macrophages drug effects, Macrophages metabolism, Macrophages, Peritoneal cytology, Macrophages, Peritoneal drug effects, Macrophages, Peritoneal metabolism, Patch-Clamp Techniques, Transient Receptor Potential Channels genetics, Transient Receptor Potential Channels metabolism, Wortmannin, rab GTP-Binding Proteins metabolism, rab5 GTP-Binding Proteins metabolism, rab7 GTP-Binding Proteins, Action Potentials drug effects, Androstadienes pharmacology, Bridged Bicyclo Compounds, Heterocyclic pharmacology, Endosomes metabolism, Thiazolidines pharmacology
- Abstract
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., (Copyright © 2017 Elsevier Ltd. All rights reserved.)
- Published
- 2017
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