Search for: All records

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. 

A far-red fluorescent protein-based indicator allowed the imaging of synaptic Zn 2+ in brain slices and awake mice. 

Potassium ion (K + ) plays a critical role as an essential electrolyte in all biological systems. Genetically-encoded fluorescent K + biosensors are promising tools to further improve our understanding of K + -dependent processes under normal and pathological conditions. Here, we report the crystal structure of a previously reported genetically-encoded fluorescent K + biosensor, GINKO1, in the K + -bound state. Using structure-guided optimization and directed evolution, we have engineered an improved K + biosensor, designated GINKO2, with higher sensitivity and specificity. We have demonstrated the utility of GINKO2 for in vivo detection and imaging of K + dynamics in multiple model organisms, including bacteria, plants, and mice.