Ratiometric Imaging for Quantification of Elevated Ca²⁺ in Neurons Using Synthetic Low-Affinity Fluorescent Probe

The availability of various calcium ion (Ca²⁺) fluorescent probes has contributed to revealing physiological events related to intracellular Ca²⁺. However, conventional probes face challenges for quantitatively and selectively visualizing high Ca²⁺ concentrations in cells induced by any stimuli, including biomolecules or electrical signal that disrupt Ca²⁺ homeostasis. In this report, we designed and synthesized a low-affinity ratiometric Ca²⁺ probe, KLCA-Fura, utilizing o-aminophenol-N,N-diacetate-O-methylene-methylphosphinate (APDAP) as a ligand, for which we recently demonstrated the suitability as a new low-affinity ligand for Ca²⁺. KLCA-Fura showed a blue shift in excitation wavelength with increasing Ca²⁺ concentration based on the intramolecular charge transfer (ICT). Its affinity for Ca²⁺ is lower than commercially available conventional Ca²⁺ probes. Furthermore, the selectivity for Ca²⁺ and the fluorescence intensity were considered sufficient to accurately detect Ca²⁺. The corresponding acetoxymethyl ester, KLCA-FuraAM, was synthesized for intracellular imaging and applied to Ca²⁺ quantification in neurons. KLCA-FuraAM enabled quantitative ratiometric monitoring of the two-step Ca²⁺ concentration increase induced by glutamate stimulation. While this two-step response was not clearly observed with a commercially available low-affinity ratiometric Ca²⁺ probe, Fura-FF, KLCA-FuraAM has demonstrated the potential to quantitatively visualize the behavior of high Ca²⁺ concentrations. The ratiometric low-affinity Ca²⁺ probe, KLCA-Fura, is expected to be a powerful tool for discovering new functions of Ca²⁺ in neurons.