Fast-Response Calmodulin-Based Fluorescent Indicators Reveal Rapid Intracellular Calcium Dynamics

Area of Science:

• Biochemistry
• Molecular Biology
• Cellular Neuroscience

Background:

• Accurate measurement of intracellular calcium (Ca2+) dynamics is crucial for understanding cellular processes.
• Existing genetically encoded calmodulin-based fluorescent indicators often exhibit kinetics that distort rapid Ca2+ signals due to saturation and integration.
• Limitations in fluorescence rise and decay kinetics of current probes hinder faithful reporting of fast Ca2+ transients.

Purpose of the Study:

• To engineer novel calmodulin-based fluorescent indicators with accelerated kinetics to accurately capture fast intracellular Ca2+ dynamics.
• To develop probes with a tunable range of Ca2+ affinities to match specific biological signals.
• To improve the fidelity of Ca2+ signal reporting in cellular and tissue imaging.

Main Methods:

• Engineered a series of genetically encoded Ca2+ indicators by modifying calmodulin-peptide interactions to accelerate kinetics.
• Weakened Ca2+-calmodulin-peptide interactions to achieve a range of Ca2+ affinities and faster response times.
• Characterized probe kinetics (rise and decay times) at 37°C and evaluated their performance in neonatal cardiac myocytes.

Main Results:

• Developed GCaMP3fast, a GCaMP3-derived probe with 40-fold faster Ca2+ kinetics (0.9 ms rise, 3.3 ms decay) compared to GCaMP3, making it the fastest reported to date.
• GCaMP3fast revealed faster Ca2+ transients in neonatal cardiac myocytes than GCaMP6f, demonstrating its ability to resolve rapid dynamics.
• GCaMP3fast exhibits a 5-fold increased two-photon fluorescence cross-section at 940 nm, enhancing its suitability for deep tissue imaging.

Conclusions:

• The engineered GCaMP3fast probe accurately reports fast intracellular Ca2+ dynamics, overcoming limitations of previous indicators.
• The strategy of tuning indicator affinity and kinetics is effective for developing next-generation calmodulin-based probes.
• GCaMP3fast provides novel insights into calmodulin's kinetic mechanism and is valuable for deep tissue Ca2+ imaging.