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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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.).
Integrated supplementary information
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.
The video shows the process of isolation of a vacuolin-enlarged lysosome from an intact HEK293 cell.
The video shows schematically the process of isolation of an enlarged lysosome from an intact cell.
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).