Calcium Green FlAsH as a genetically targeted small-molecule calcium indicator

Area of Science:

• Cellular Biology
• Biochemistry
• Molecular Imaging

Background:

• Intracellular calcium (Ca2+) is a critical second messenger regulating diverse cellular functions.
• Existing Ca2+ detection methods often lack the spatial and temporal resolution to capture rapid, localized signaling events.
• Submicron and submillisecond Ca2+ variations are crucial for understanding protein regulation and cellular processes.

Purpose of the Study:

• To develop a novel, high-resolution Ca2+ indicator for probing localized intracellular Ca2+ dynamics.
• To investigate Ca2+ signaling at genetically targeted proteins and cellular structures, such as gap junctions and L-type calcium channels.

Main Methods:

• Creation of Calcium Green FlAsH (CaGF), a ~1-kDa biarsenical Ca2+ indicator.
• Attachment of CaGF to tetracysteine motifs for genetically targeted protein labeling.
• Utilized total internal reflection microscopy to visualize CaGF-labeled proteins and Ca2+ transients in HeLa cells.

Main Results:

• CaGF exhibits a tenfold increase in fluorescence upon Ca2+ binding, with a Kd of ~100 μM, <1-ms kinetics, and good Mg2+ rejection.
• CaGF successfully labeled tetracysteine-tagged connexin 43 in gap junctions, reporting Ca2+ waves after injury.
• Observed rapid, depolarization-evoked Ca2+ transients at L-type calcium channels, revealing spatial non-uniformity in channel opening probability and Ca2+ domains.

Conclusions:

• CaGF is a sensitive and fast Ca2+ indicator capable of reporting highly localized, rapid [Ca2+] dynamics.
• The indicator allows for the study of Ca2+ signaling in proximity to specific proteins and in distinct subcellular compartments.
• Findings highlight the complex spatial organization of Ca2+ signaling, particularly around ion channels.