Brief Guide to Patch-Clamp Current Measurements in Organelle Membranes

Abstract

The standard giga-seal patch-clamp techniques opened the way to monitor ion-channel activity in the native organelle membrane in situ. One has to have an organelle preparation, covered with the intact mem-brane carrying rightly oriented channel proteins and suitable for proper patch-clamping. Here we deal with the intact " native " organelle, the nucleus with an inner and outer nucleic membrane (nuclear envelope), and mitochondria without the outer membrane but an intact inner membrane (mitoplast). We collect them by rupturing the cell using a variety of procedures including cell homogenization, cell swelling with a hypotonic solution, cell shaking in a Na citrate solution, and their appropriate combination, usually modified by individual laboratories. After homogenization of cells or a piece of tissue, we can collect mitochondria (1) from any type of cell with the standard sucrose density-gradient centrifugation procedure. It is a routine procedure and has no immediate problems. Rupturing the outer mitochon-drial membrane by repeating hypotonic and hypertonic solution exposures, we have mitoplasts suitable for patch-clamping.