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

• Published in: Nature protocols Vol. 12; no. 8; pp. 1639 - 1658
• Main Authors: Chen, Cheng-Chang, Cang, Chunlei, Fenske, Stefanie, Butz, Elisabeth, Chao, Yu-Kai, Biel, Martin, Ren, Dejian, Wahl-Schott, Christian, Grimm, Christian
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 01.08.2017; Nature Publishing Group
• Subjects: 631/1647/1453/1970; 631/1647/1453/2206; 631/45/269; 631/80/642; Analysis; Analytical Chemistry; Animals; Biological Techniques; Cell membranes; Cells, Cultured; Compartments; Computational Biology/Bioinformatics; Data analysis; Data processing; Development and progression; Disorders; Electrophysiology; Endosomes; Endosomes - metabolism; Enlargement; Humans; Infectious diseases; Intracellular; Ion channels; Ion Channels - metabolism; Ions; Life Sciences; Lysosomal storage diseases; Lysosomes; Lysosomes - metabolism; Membranes; Methods; Mice; Microarrays; Neurodegenerative diseases; Neurological diseases; Organelles; Organic Chemistry; Patch-Clamp Techniques - methods; Potassium; Proteomics; protocol; Therapeutic targets; Visual control
• ISSN: 1754-2189; 1750-2799
• DOI: 10.1038/nprot.2017.036

Chen, Cang and colleagues describe how to characterize intracellular ion channels using a manual patch-clamp technique on enlarged organelles such as endolysosomes. 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.