Preparation of Giant Escherichia coli spheroplasts for Electrophysiological Recordings

Prokaryotic ion channels have been instrumental in furthering our understanding of many fundamental aspects of ion channels' structure and function. However, characterizing the biophysical properties of a prokaryotic ion channel in a native membrane system using patch-clamp electrophysiology is technically challenging. Patch-clamp is regarded as a gold standard technique to study ion channel properties in both native and heterologous expression systems. The presence of a cell wall and the small size of bacterial cells makes it impossible to directly patch clamp using microelectrodes. Here, we describe a method for the preparation of giant E. coli spheroplasts in order to investigate the electrophysiological properties of bacterial cell membranes. Spheroplasts are formed by first inhibiting bacterial cell wall synthesis, followed by enzymatic digestion of the outer cell wall in the presence of a permeabilizing agent. This protocol can be used to characterize the function of any heterologous ion channels or ion transporters expressed in E. coli membranes.