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Patch-clamp technique to characterize ion channels in enlarged individual endolysosomes

Nature Protocols volume 12, pages 16391658 (2017) | Download Citation

Abstract

According to proteomics analyses, more than 70 different ion channels and transporters are harbored in membranes of intracellular compartments such as endosomes and lysosomes. Malfunctioning of these channels has been implicated in human diseases such as lysosomal storage disorders, neurodegenerative diseases and metabolic pathologies, as well as in the progression of certain infectious diseases. As a consequence, these channels have engendered very high interest as future drug targets. Detailed electrophysiological characterization of intracellular ion channels is lacking, mainly because standard methods to analyze plasma membrane ion channels, such as the patch-clamp technique, are not readily applicable to intracellular organelles. Here we present a protocol detailing how to implement a manual patch-clamp technique for endolysosomal compartments. In contrast to the alternatively used planar endolysosomal patch-clamp technique, this method is a visually controlled, direct patch-clamp technique similar to conventional patch-clamping. The protocol assumes basic knowledge and experience with patch-clamp methods. Implementation of the method requires up to 1 week, and material preparation takes 2–4 d. An individual experiment (i.e., measurement of channel currents across the endolysosomal membrane), including control experiments, can be completed within 1 h. This excludes the time for endolysosome enlargement, which takes between 1 and 48 h, depending on the approach and cell type used. Data analysis requires an additional hour.

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Acknowledgements

This work was supported, in part, by funding from the German Research Foundation (SFB/TRR152 TP04 to C.G., TP06 to C.W.-S., and TP12 to M.B., as well as SFB870 TP05 to C.W.-S., TP10 to M.B., and TP15 to C.W.-S.).

Author information

Author notes

    • Cheng-Chang Chen
    •  & Chunlei Cang

    These authors contributed equally to this work.

Affiliations

  1. Department of Pharmacy – Center for Drug Research and Center for Integrated Protein Science Munich (CIPSM), Ludwig-Maximilians-Universität München, München, Germany.

    • Cheng-Chang Chen
    • , Stefanie Fenske
    • , Elisabeth Butz
    • , Yu-Kai Chao
    • , Martin Biel
    • , Christian Wahl-Schott
    •  & Christian Grimm
  2. School of Life Sciences, University of Science & Technology of China, Hefei, China.

    • Chunlei Cang
  3. Department of Biology, University of Pennsylvania, Philadelphia, Pennsylvania, USA.

    • Dejian Ren

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Contributions

C.-C.C. and C.C. developed and performed endolysosomal patch-clamp experiments. C.-C.C., C.C., E.B. and Y.-K.C. designed, collected and/or analyzed data. C.-C.C., C.C., S.F., D.R., C.G., C.W.-S. and M.B. designed the study and edited the manuscript. All the authors discussed the results and commented on the manuscript.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to Christian Wahl-Schott or Christian Grimm.

Integrated supplementary information

Supplementary information

PDF files

  1. 1.

    Supplementary Text and Figures

    Supplementary Figures 1 and 2, the Supplementary Note, and Supplementary Table 1.

Videos

  1. 1.

    Vacuolin-mediated enlargement of LAMP1-positive vesicles in MEF cells.

    The video shows the enlargement of LAMP1-positive vesicles in MEF cells over a time interval of 50 min. Lamp1-GFP stably transfected MEF cells were treated with 3 μM vacuolin and incubated for 30 min at 37 °C in a 5% CO2 atmosphere prior to imaging. For imaging, cells were transferred to a microscope equipped with a climate imaging chamber (5% CO2, 37 °C) and incubated in imaging buffer containing 3 μM vacuolin during experimentation. Images were taken in 30-s intervals. Note that a subpopulation of LAMP1-positive vesicles enlarge up to a size enabling patch-clamp experimentation. Scale bar, 10 μm.

  2. 2.

    Live enlarged lysosome isolation procedure

    The video shows the process of isolation of a vacuolin-enlarged lysosome from an intact HEK293 cell.

  3. 3.

    Enlarged lysosome isolation procedure

    The video shows schematically the process of isolation of an enlarged lysosome from an intact cell.

  4. 4.

    Pipette preparation procedure

    The video shows the process of pipette preparation for endolysosomal patch-clamp experimentation. The protocol is optimized for the following equipment: micropipette (borosilicate glass with filament, fire polished, O.D. 1.5 mm, I.D. 0.75 mm, length 10 cm; Sutter Instrument, cat. no. BF150-75-10); P-97 Flaming/Brown-type micropipette puller (Sutter Instrument, cat. no. FT330B); 3.0-mm wide-trough filament (World Precision Instruments); MF-830 microforge with platinum heater (Narishige).

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DOI

https://doi.org/10.1038/nprot.2017.036

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