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

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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Figure 1
Figure 2: Enlargement of intracellular organelles in HEK293 cells upon treatment.
Figure 3: Equipment used for the fabrication of optimal patch pipette tips.
Figure 4: Fire-polishing of patch-clamp pipettes.
Figure 5: Dissection procedure for preparation of individual enlarged endolysosomes from a single cell.
Figure 6: Schematic showing ion channels that have been measured with the endolysosomal patch-clamp technique.
Figure 7: Series of images illustrating the continuous process of isolation of a vacuolin-1-enlarged endolysosomal vesicle in a HEK293 cell (2-h treatment) with an 'isolation pipette' and gigaseal formation with the 'recording pipette'.
Figure 8: Currents for calculation of pipette resistance, seal resistance, series resistance and cell capacitance.
Figure 9: Patch-clamp configurations for single-lysosome recordings.
Figure 10: Representative whole-endolysosome recordings.
Figure 11: Representative current-clamp recording from a vacuolin-1 (5 μM overnight)-enlarged endolysosome isolated from a cortical astrocyte showing steady-state value and dynamic changes of lysosomal membrane potential.
Figure 12: Single-channel recordings.

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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

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Authors

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.

Corresponding authors

Correspondence to Christian Wahl-Schott or Christian Grimm.

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Competing interests

The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Vacuolin-mediated enlargement of LAMP1-positive vesicles in MEF cells.

The image series shows enlargement of LAMP1-positive vesicles in MEF cells after defined time intervals. Images were taken from Video 1.

Supplementary Figure 2 Cartoon showing patch-clamp configurations.

a, voltage clamp experiment in whole-cell configuration (upper cartoon): In a whole cell patch clamp experiment the extracellular surface faces the bath solution which is connected to the reference electrode (ground). The potential of the bath solution and the extracellular surface of the cell is 0 mV. The patch clamp pipette solution is connected to the cytosolic side of the cell. To clamp a cell at a membrane potential of e.g. -90 mV, a negative potential of – 90 mV at the headstage input with respect to ground needs to be delivered; the potential inside the patch clamp pipette (= command voltage) needs to be set to -90 mV. Thus, the membrane potential is directly proportional to the command voltage. The upper cartoon shows cations (e.g. Na+) flowing from the outside into the cell (cytosol). A flow of cations (here Na+) into the cells represents a depolarizing current. In voltage clamp experiments, this current is clamped by the amplifier by a flow of cations into the headstage, which is defined as a negative current. Thus, inward membrane currents correspond to inward command currents into the headstage. In whole cell patch clamp command currents and membrane currents are proportionally related. b, voltage clamp experiment in whole endolysosomal configuration (lower cartoon): Once the endolysosome is released from the cell, the membrane surface that was originally facing the cytosol is exposed to the bath solution, which is connected to the reference electrode (ground). Now the potential on this membrane surface is 0 mV (bath potential). The pipette is still connected to the luminal surface of the endolysosomal membrane. For a membrane potential of e.g. Vm = -90 mV, the potential inside the pipette must be adjusted to the more depolarized value of +90 mV (top). Thus, the potential across the endolysosomal membrane is inversely proportional to the command potential. In order to match the conditions outlined for whole cell recordings, the command voltage needs to be inverted. The lower cartoon shows cations (e.g. Na+) flowing from the endolysosomal lumen into the bath solution (corresponds to the cytosol). In voltage clamp experiments, this current is compensated by a flow of cations out of the headstage into the pipette and subsequently out of the pipette tip, which is defined as a positive current. Therefore a positive command current corresponds to a membrane current carried by cations flowing from the lysosomal lumen into the cytosol. In whole endolysosomal patch clamp command currents and membrane currents are inversely related. In order to match the conventions that flow of cations into the cytosol represents an inward current the command current needs to be inverted.

Summary: Flow of Na+ into the cytosol corresponds to a negative command current in a and a positive command current in b. To match conventions that the flow of cations into the cytosol is defined as a negative current the command current in b is inverted. In order to clamp the membrane voltage to e.g. -90 mV cytosol negative with respect to the extracellular side, a negative command voltage is applied in a while a positive command voltage is applied in b. To match conventions that membrane voltage Vm is given as the difference (Δ) of the cytosolic voltage Vcytosol minus the Vnon-cytosol voltage in the compartment on the other side of the membrane under investigation (extracellular side in the whole cell recording) voltage is inverted in b.

Top rows in a and b (right part of the upper panel): command voltage (left) at headstage input and command current (right) into or out of the headstage input. Definition: The flow of positive current out of the headstage into the patch clamp pipette and out of the pipette tip is termed positive current. A positive potential means a positive voltage at the headstage input with respect to ground (for details see e.g. The Axon Guide for Electrophysiology & Biophysics Laboratory Techniques). Bottom rows in a and b (right): Current and voltages are given with respect to the cytosol. Currents flowing into the cytosol are negative currents, membrane voltages are given as difference of cytosolic voltage minus the voltage in the non-cytosolic compartment (for details see e.g. The Axon Guide for Electrophysiology & Biophysics Laboratory Techniques).

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1 and 2, the Supplementary Note, and Supplementary Table 1. (PDF 431 kb)

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. (MP4 3759 kb)

Live enlarged lysosome isolation procedure

The video shows the process of isolation of a vacuolin-enlarged lysosome from an intact HEK293 cell. (MP4 15712 kb)

Enlarged lysosome isolation procedure

The video shows schematically the process of isolation of an enlarged lysosome from an intact cell. (MP4 2858 kb)

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). (MP4 8020 kb)

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Chen, CC., Cang, C., Fenske, S. et al. Patch-clamp technique to characterize ion channels in enlarged individual endolysosomes. Nat Protoc 12, 1639–1658 (2017). https://doi.org/10.1038/nprot.2017.036

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