Tuning the sensitivity of genetically encoded fluorescent potassium indicators through structure-guided and genome mining strategies

Abstract

AbstractGenetically encoded potassium indicators lack optimal binding affinity for monitoring intracellular dynamics in mammalian cells. Through structure-guided design and genome mining of potassium binding proteins, we developed green fluorescent potassium indicators with a broad range of binding affinities. KRaION1, based on the insertion of a potassium binding protein (Ec-Kbp) into the fluorescent protein mNeonGreen, exhibits an isotonically measured Kd of 69±10 (mM; mean ± standard deviation used throughout). We identified Ec-Kbp’s binding site using NMR spectroscopy to detect protein-thallium scalar couplings and refined the structure of Ec-Kbp in its potassium-bound state. Guided by this structure, we modified KRaION1, yielding KRaION2, which exhibits an isotonically measured Kd of 96±9 (mM). We identified four Ec-Kbp homologs as potassium binding proteins, which yielded indicators with isotonically measured binding affinities in the 39-112 (mM) range. KRaIONs expressed and functioned in HeLa cells, but exhibited lower Kd values, which were mirrored by lower Kd values measured in vitro when holding sodium constant. Thus, potassium indicator Kd may need to be evaluated in the context of a given experimental goal.