Neuronal Calcium Recording with an Engineered TEV Protease.

Area of Science:

• Neuroscience
• Biochemistry
• Molecular Biology

Background:

• Transient calcium (Ca2+) spikes are crucial for neural signaling, but their visualization across large brain regions or thick specimens is challenging.
• Existing Ca2+ reporters often capture transient signals, limiting their utility in complex biological systems.
• Stable integrators of Ca2+ offer a promising alternative for studying neural activity in difficult contexts.

Discussion:

• An engineered Ca2+-sensing enzyme, SCANR (Split TEV, Ca2+ Activated Neuron Recorder), was developed using a split Tobacco Etch Virus (TEV) protease system.
• Each half of the TEV protease is linked to a Ca2+-binding domain (calmodulin or M13).
• SCANR remains inactive until cellular Ca2+ spikes trigger its reconstitution and enzymatic activity.

Key Insights:

• SCANR becomes catalytically active upon binding Ca2+, leading to the turnover of a caged, genetically encoded reporter substrate.
• A functional Ca2+-sensing split TEV enzyme was identified from a library of chimeras.
• The SCANR system was successfully deployed in primary rat hippocampal neurons, demonstrating its practical application.

Outlook:

• SCANR provides a novel tool for visualizing neural signaling dynamics with improved stability and sensitivity.
• This technology has the potential to advance our understanding of brain function in various physiological and pathological conditions.
• Further applications of SCANR may include in vivo imaging and studying neural circuits in complex tissues.