Enhanced Archaerhodopsin Fluorescent Protein Voltage Indicators.

Area of Science:

• Neuroscience
• Molecular Biology
• Biophysics

Background:

• Imaging voltage dynamics in specific neuronal populations is a key neuroscience goal.
• Genetically encoded fluorescent voltage indicators, like Archaerhodopsin (Arch), show promise for monitoring neural activity.
• Existing Arch-based sensors have limitations, including slow kinetics or unwanted photocurrents.

Purpose of the Study:

• To develop improved Arch-derived voltage sensors for enhanced neural membrane localization and function.
• To engineer Arch mutants with faster kinetics, greater dynamic range, and no photocurrent during imaging.
• To quantitatively assess the spike detection fidelity of novel voltage sensors.

Main Methods:

• Engineered Arch mutants (Arch-EEN, Arch-EEQ) with modified sequences and added trafficking signals.
• Benchmarking sensor performance using a signal detection theoretic framework.
• Analysis incorporated experimental photon shot noise and optical waveforms of action potentials.

Main Results:

• Novel Arch-derived sensors exhibit enhanced localization to the neural membrane.
• Arch-EEN and Arch-EEQ demonstrate faster kinetics and higher fluorescence dynamic range than Arch-D95N.
• The new sensors achieved nearly a three-fold improvement in spike detection fidelity compared to Arch-D95N.

Conclusions:

• Genetically encoded voltage sensors based on Arch can be significantly improved through targeted mutations and trafficking sequences.
• These enhanced sensors offer superior performance for imaging neuronal voltage dynamics, particularly for detecting single action potentials.
• The developed sensors represent a significant advancement for studying neural circuits in various biological contexts.