Ion Transport Activity Assay for Microbial Rhodopsin Expressed in Escherichia coli Cells

Microbial rhodopsins have diverse functions, including roles as light-driven ion pumps, light-gated ion channels, photosensors, and light-regulated enzymes. As the number of rhodopsin-like genes identified has increased in recent years, so has the requirement for rapid identification of their functions. The patch-clamp method is often used to investigate the ion transport mechanism of microbial rhodopsins in mammalian cells; however, this requires a dedicated system and advanced techniques. The ion transport assay using the Escherichia coli expression system described here evaluates the ion transport capacity by monitoring the pH change in E. coli suspensions; if the target rhodopsin has a light-dependent ion transport activity, a light-dependent pH change is observed. The pH increase or decrease corresponds to proton release from the cell or proton uptake into the cell, respectively. This method can be used to evaluate ion transport capacity in a high-throughput manner using a combination of general-purpose equipment and common techniques. Graphic abstract: Schematic diagram of the ion transport assay in rhodopsin-expressing E. coli cells.