Patch-Clamp Recording of the Activity of Ion Channels in the Inner Mitochondrial Membrane.

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.