Tuning the Sensitivity of Genetically Encoded Fluorescent Potassium Indicators through Structure-Guided and Genome Mining Strategies

Genetically encoded potassium indicators lack optimal binding affinity for monitoring intracellular dynamics inmammalian cells. Through structure-guided design and genomemining of potassium binding proteins, we developed greenfluorescent potassium indicators with a broad range of bindingaffinities. KRaION1 (K+ratiometric indicator for optical imagingbased on mNeonGreen 1), based on the insertion of a potassiumbinding protein, Kbp, fromE. coli(Ec-Kbp) into thefluorescentprotein mNeonGreen, exhibits an isotonically measuredKdof 69 +/- 10 mM (mean +/- standard deviation used throughout). Weidentified Ec-Kbp's binding site using NMR spectroscopy to detectprotein-thallium scalar couplings and refined the structure of Ec-Kbp in its potassium-bound state. Guided by this structure, we modified KRaION1, yielding KRaION1/D9N and KRaION2, whichexhibit isotonically measuredKd's of 138 +/- 21 and 96 +/- 9 mM. We identified four Ec-Kbp homologues as potassium bindingproteins, which yielded indicators with isotonically measured binding affinities in the 39-112 mM range. KRaIONs functioned inHeLa cells, but theKdvalues differed from the isotonically measured case. We found that, by tuning the experimental conditions,Kdvalues could be obtained that were consistentin vitroandin vivo. We thus recommend characterizing potassium indicatorKdin the physiological context of interest before application