A sensitive and specific genetically encoded potassium ion biosensor for in vivo applications across the tree of life

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.