Abstract
The availability of various calcium ion (Ca(2+)) fluorescent probes has contributed to revealing physiological events related to intracellular Ca(2+). However, conventional probes face challenges for quantitatively and selectively visualizing high Ca(2+) concentrations in cells induced by any stimuli, including biomolecules or electrical signal that disrupt Ca(2+) homeostasis. In this report, we designed and synthesized a low-affinity ratiometric Ca(2+) 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 Ca(2+). KLCA-Fura showed a blue shift in excitation wavelength with increasing Ca(2+) concentration based on the intramolecular charge transfer (ICT). Its affinity for Ca(2+) is lower than commercially available conventional Ca(2+) probes. Furthermore, the selectivity for Ca(2+) and the fluorescence intensity were considered sufficient to accurately detect Ca(2+). The corresponding acetoxymethyl ester, KLCA-FuraAM, was synthesized for intracellular imaging and applied to Ca(2+) quantification in neurons. KLCA-FuraAM enabled quantitative ratiometric monitoring of the two-step Ca(2+) concentration increase induced by glutamate stimulation. While this two-step response was not clearly observed with a commercially available low-affinity ratiometric Ca(2+) probe, Fura-FF, KLCA-FuraAM has demonstrated the potential to quantitatively visualize the behavior of high Ca(2+) concentrations. The ratiometric low-affinity Ca(2+) probe, KLCA-Fura, is expected to be a powerful tool for discovering new functions of Ca(2+) in neurons.