Unlocking chloride sensing in the red at physiological pH with a fluorescent rhodopsin-based host

Chloride is a vital ion for all forms of life. Nature has evolved elegant supramolecular machines to facilitate chloride transport across cell membranes. Protein-based fluorescent biosensors can enable researchers to intercept and monitor chloride in living cells but remain underdeveloped. Here, we demonstrate how this can be achieved through the introduction of a single point mutation in an engineered, microbial rhodopsin from the archaebacterium Halorubrum sodomense resulting in ChloRED-1-CFP. This membrane-bound host is a far-red emitting, ratiometric sensor that provides a reversible readout of chloride in live bacteria at physiological pH, setting the stage to investigate the roles of chloride in a wide range of biological contexts.