Potassium is the most abundant intracellular
metal in the body, playing vital roles in regulating intracellular fluid volume,
nutrient transport, and cell-to-cell communication through nerve and muscle
contraction. On the other hand, aberrant alterations in K<sup>+</sup>
homeostasis contribute to a diverse array of diseases spanning cardiovascular
and neurological disorders to diabetes to kidney disease to cancer. Owing to
the large differences in intracellular versus extracellular K<sup>+</sup> concentrations
([K<sup>+</sup>]<sub>intra</sub> = 150 mM, [K<sup>+</sup>]<sub>extra</sub> =
3-5 mM), an unmet need for studies of K<sup>+</sup> physiology and pathology
remains a relative dearth of methods to reliably measure dynamic changes in
intracellular K<sup>+</sup> in biological specimens that meet the dual
challenges of low affinity and high selectivity for K<sup>+</sup>, particularly
over Na<sup>+</sup>, as currently available fluorescent K<sup>+</sup> sensors are
largely optimized with high-affinity receptors that are more amenable for
extracellular K<sup>+</sup> detection. We report the design, synthesis, and
biological evaluation of Ratiometric Potassium Sensor 1 (<b>RPS-1</b>), a dual-fluorophore
sensor that enables ratiometric fluorescence imaging of intracellular potassium
in living systems. <b>RPS-1</b> links a potassium-responsive fluorescent sensor
fragment (<b>PS525</b>) with a low-affinity, high-selectivity crown ether receptor
for K<sup>+</sup> to a potassium-insensitive reference fluorophore (<b>Coumarin
343</b>) as an internal calibration standard through ester bonds. Upon intracellular
delivery, esterase-directed cleavage splits these two dyes into separate
fragments to enable ratiometric detection of K<sup>+</sup>. <b>RPS-1</b>
responds to K<sup>+</sup> in aqueous buffer with high selectivity over
competing metal ions and is sensitive to potassium ions at steady-state intracellular
levels and can respond to decreases or increases from that basal set point.
Moreover, <b>RPS-1</b> was applied for comparative screening of K<sup>+</sup>
pools across a panel of different cancer cell lines, revealing elevations in basal
intracellular K<sup>+</sup> in metastatic breast cancer cell lines vs normal
breast cells. This work provides a unique chemical tool for the study of
intracellular potassium dynamics and a starting point for the design of other
ratiometric fluorescent sensors based on two-fluorophore approaches that do not
rely on FRET or related energy transfer designs.