Sensitive red fluorescent indicators for real-time visualization of potassium ion dynamics in vivo.

Potassium ion (K+) dynamics are vital for various biological processes. However, the limited availability of detection tools for tracking intracellular and extracellular K+ has impeded a comprehensive understanding of the physiological roles of K+ in intact biological systems. In this study, we developed two novel red genetically encoded potassium indicators (RGEPOs), RGEPO1 and RGEPO2, through a combination of directed evolution in Escherichia coli and subsequent optimization in mammalian cells. RGEPO1, targeted to the extracellular membrane, and RGEPO2, localized in the cytoplasm, exhibited positive K+-specific fluorescence response with affinities of 2.4 and 43.3 mM in HEK293FT cells, respectively. We employed RGEPOs for real-time monitoring of subsecond K+ dynamics in cultured neurons, astrocytes, acute brain slices, and the awake mouse in both intracellular and extracellular environments. Using RGEPOs, we were able, for the first time, to visualize intracellular and extracellular potassium transients during seizures in the brains of awake mice. Furthermore, molecular dynamics simulations provided new insights into the potassium-binding mechanisms of RGEPO1 and RGEPO2, revealing distinct K+-binding pockets and structural features. Thus, RGEPOs represent a significant advancement in potassium imaging, providing enhanced tools for real-time visualization of K+ dynamics in various cell types and cellular environments.