Analyzing plant mechanosensitive ion channels expressed in giant E. coli spheroplasts by single-channel patch-clamp electrophysiology

Plants possess numerous ion channels that respond to a range of stimuli, including small molecules, transmembrane voltage, and mechanical force. Many in the latter category, known as mechanosensitive (MS) ion channels, open directly in response to increases in lateral membrane tension. One of the most effective techniques for characterizing ion channel properties is patch-clamp electrophysiology, in which the current through a section of membrane containing ion channels is measured. For MS channels, this technique enables the measurement of key channel properties such as tension sensitivity, conductance, and ion selectivity. These characteristics, along with the phenotypes of genetic mutants, can help reveal the physiological roles of a particular MS channel. In this protocol, we provide detailed instructions on how to study MS ion channels using single-channel patch-clamp electrophysiology in giant E. coli spheroplasts. We first present an optimized method for preparing giant spheroplasts, then describe how to measure MS channel activity using patch-clamp electrophysiology and analyze the resulting data. We also provide recommended equipment lists, setup schematics, and useful conventions.