Department of Physics and Biophysics, Institute of Biology, Warsaw University of Life Sciences-SGGW, Warsaw, Poland.
Laboratory of Intracellular Ion Channels, Nencki Institute of Experimental Biology, Warsaw, Poland.
Methods Mol Biol. 2021;2276:235-248. doi: 10.1007/978-1-0716-1266-8_18.
Mitochondria are intracellular organelles, which play a crucial role in the generation of ATP. Mitochondria are surrounded by a double membrane, consisting of a smooth outer membrane (OMM) and a markedly folded inner mitochondrial membrane (IMM). Mitochondrion that has been stripped of its outer membrane, leaving the inner membrane intact is called mitoplast. There is a number of different transport proteins located in the inner mitochondrial membrane including ion channels that mediate fluxes of potassium, calcium, and chloride ions. These channels regulate the mitochondrial membrane potential, respiration, and production of reactive oxygen species. The stability of mitoplasts offers the possibility of measuring the activity of ion channels from IMM using the patch-clamp technique. Electrophysiological measurements of currents through ion channels in the IMM permit discovery of unique properties of these channels with the aim of new specific pharmacological therapies. In this chapter, we describe the isolation of mitochondria, preparation of mitoplast for patch-clamp recordings and single-mitoplast PCR experiments, which can be helpful in mastering the technique of recording the activity of mitochondrial ion channels.
线粒体是细胞内的细胞器，在 ATP 的生成中起着至关重要的作用。线粒体被双层膜包围，由光滑的外膜（OMM）和明显折叠的内线粒体膜（IMM）组成。已经去除其外膜而保留内膜完整的线粒体称为线粒体体。在内线粒体膜上有许多不同的转运蛋白，包括介导钾、钙和氯离子通量的离子通道。这些通道调节线粒体膜电位、呼吸和活性氧物质的产生。线粒体体的稳定性提供了使用膜片钳技术从 IMM 测量离子通道活性的可能性。通过 IMM 中的离子通道的电生理测量允许发现这些通道的独特性质，目的是进行新的特定药理学治疗。在本章中，我们描述了线粒体的分离、线粒体体用于膜片钳记录和单线粒体体 PCR 实验的制备，这有助于掌握记录线粒体离子通道活性的技术。
