Localizable fluorescent metal ion indicators with tunable colors

Elucidating the role of metal ion homeostasis in physiological and pathological processes requires detection tools with high sensitivity and selectivity, spectral versatility, and precise subcellular localization. Here, we introduce a modular strategy for generating fluorescent metal ion indicators which are comprised of a sulfonamide-functionalized chelator, a rhodamine derivative and a ligand for bioconjugation to self-labeling proteins. With a concise three-step synthesis, the design allows tuning of both the emission color and ligand specificity of the resulting indicators. Specifically, we developed a family of bright, color-tunable potassium indicators which can be selectively coupled to intra- or extracellular HaloTag and SNAP-tag fusion proteins. The increase in fluorescence upon binding to HaloTag or SNAP-tag fluorogenicity enabled wash-free live-cell imaging. The localized potassium indicators enabled the detection of dynamic potassium efflux in rat hippocampal neurons upon glutamate stimulation. Our work thus establishes a versatile platform for the generation of localizable fluorescent metal ion indicators and opens up new avenues for live-cell potassium sensing.