Two-pore channels control Ebola virus host cell entry and are drug targets for disease treatment. High susceptibility to fatty liver disease in two-pore channel 2-deficient mice. Role of TRPML and two-pore channels in endolysosomal cation homeostasis. PI(3,5)P(2) controls membrane trafficking by direct activation of mucolipin Ca(2+) release channels in the endolysosome. TMEM175 is an organelle K(+) channel regulating lysosomal function.
The type IV mucolipidosis-associated protein TRPML1 is an endolysosomal iron release channel. mTOR regulates lysosomal ATP-sensitive two-pore Na(+) channels to adapt to metabolic state. Data analysis requires an additional hour.Ĭang, C. This excludes the time for endolysosome enlargement, which takes between 1 and 48 h, depending on the approach and cell type used. An individual experiment (i.e., measurement of channel currents across the endolysosomal membrane), including control experiments, can be completed within 1 h.
Implementation of the method requires up to 1 week, and material preparation takes ∼2–4 d. The protocol assumes basic knowledge and experience with patch-clamp methods. 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. Here we present a protocol detailing how to implement a manual patch-clamp technique for endolysosomal compartments. 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. As a consequence, these channels have engendered very high interest as future drug targets. 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. According to proteomics analyses, more than 70 different ion channels and transporters are harbored in membranes of intracellular compartments such as endosomes and lysosomes.
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