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

Yuzuka Kuronuma1, Yutaka Shindo2,3, Rei Kumada1

  • 1Department of Applied Chemistry, Faculty of Science and Technology, Keio University, 3-14-1 Hiyoshi, Kohoku-ku, Yokohama, Kanagawa223-8522, Japan.

ACS Chemical Neuroscience
|January 31, 2025
PubMed
Summary

A new low-affinity calcium ion (Ca2+) probe, KLCA-FuraAM, enables quantitative imaging of high intracellular Ca2+ concentrations. This probe accurately visualizes neuronal responses to glutamate, outperforming existing tools.

Keywords:
calcium ionfluorescenceintracellular imaginglow-affinity proberatiometric measurement

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Area of Science:

  • Biochemistry and Molecular Biology
  • Neuroscience
  • Chemical Biology

Background:

  • Intracellular calcium ion (Ca2+) dynamics are crucial for physiological events.
  • Conventional Ca2+ probes struggle with quantitative and selective imaging of high Ca2+ concentrations.
  • Disruptions in Ca2+ homeostasis by various stimuli necessitate improved detection methods.

Purpose of the Study:

  • To design and synthesize a novel low-affinity ratiometric Ca2+ probe for accurate high Ca2+ detection.
  • To evaluate the probe's performance in quantitative intracellular Ca2+ imaging, particularly in neurons.
  • To assess the probe's ability to visualize complex Ca2+ signaling events.

Main Methods:

  • Synthesis of a low-affinity ratiometric Ca2+ probe, KLCA-Fura, using an o-aminophenol-N,N-diacetate-O-methylene-methylphosphinate (APDAP) ligand.
  • Preparation of the acetoxymethyl ester derivative, KLCA-FuraAM, for intracellular imaging.
  • Application of KLCA-FuraAM for quantitative ratiometric monitoring of Ca2+ in glutamate-stimulated neurons.

Main Results:

  • KLCA-Fura exhibits a blue shift in excitation wavelength with increasing Ca2+ concentration due to intramolecular charge transfer (ICT).
  • KLCA-FuraAM demonstrated sufficient selectivity and fluorescence intensity for accurate Ca2+ detection.
  • KLCA-FuraAM successfully quantified a two-step Ca2+ increase in neurons upon glutamate stimulation, a response not clearly observed with Fura-FF.

Conclusions:

  • KLCA-FuraAM is a potent tool for quantitatively visualizing high intracellular Ca2+ concentrations.
  • The probe enables accurate monitoring of complex, stimulus-induced Ca2+ dynamics in neurons.
  • KLCA-Fura represents a significant advancement for exploring novel Ca2+ functions in neuronal physiology.