Characterization of the ER-Targeted Low Affinity Ca(2+) Probe D4ER

Area of Science:

• Cell Biology
• Molecular Biology
• Neuroscience

Background:

• Calcium ion (Ca2+) is a critical intracellular messenger affecting cellular functions.
• The endoplasmic reticulum (ER) is the primary intracellular Ca2+ store, with lumenal concentrations exceeding 1 mM.
• Existing ER-targeted Ca2+ sensors have limitations, including non-ratiometric signals, calibration difficulties, and unsuitable Ca2+ affinity for ER measurements.

Purpose of the Study:

• To characterize a novel ER-targeted, Förster resonance energy transfer (FRET)-based Ca2+ sensor, D4ER.
• To assess D4ER's suitability for monitoring Ca2+ concentration ([Ca2+]) variations within the ER lumen.
• To evaluate ER Ca2+ homeostasis in cells expressing mutated Presenilin 2 (PS2) using D4ER.

Main Methods:

• Development and characterization of the D4ER Cameleon probe.
• Utilizing FRET-based imaging to monitor ER Ca2+ dynamics.
• Measuring resting ER Ca2+ levels ([Ca2+]ER) in cells with and without mutated PS2.

Main Results:

• D4ER exhibits suitable Ca2+ affinity and dynamic range for ER lumenal measurements.
• The probe provides ratiometric signals, facilitating accurate calibration and reducing artifacts.
• A conspicuous reduction in ER Ca2+ content was detected in cells expressing mutated PS2 compared to controls.

Conclusions:

• D4ER is a valuable tool for precise measurement of ER Ca2+ concentrations.
• The probe's characteristics overcome limitations of previous Ca2+ sensors.
• D4ER facilitates the study of ER Ca2+ dysregulation in diseases such as Alzheimer's disease.