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Small Molecules for Early Endosome-Specific Patch Clamping.
- Source :
-
Cell chemical biology [Cell Chem Biol] 2017 Jul 20; Vol. 24 (7), pp. 907-916.e4. - Publication Year :
- 2017
-
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.<br /> (Copyright © 2017 Elsevier Ltd. All rights reserved.)
- 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
Subjects
Details
- Language :
- English
- ISSN :
- 2451-9448
- Volume :
- 24
- Issue :
- 7
- Database :
- MEDLINE
- Journal :
- Cell chemical biology
- Publication Type :
- Academic Journal
- Accession number :
- 28732201
- Full Text :
- https://doi.org/10.1016/j.chembiol.2017.05.025