Blue-shifted ancyromonad channelrhodopsins for multiplex optogenetics.

Area of Science:

• Optogenetics
• Molecular Biology
• Neuroscience

Background:

• Channelrhodopsins (ChRs) are essential optogenetic tools for controlling neuronal and cardiomyocyte activity.
• Developing spectrally distinct ChRs for multiplex applications is challenging without compromising channel function.

Purpose of the Study:

• To identify and characterize novel, blue-shifted channelrhodopsins from ancyromonads.
• To expand the optogenetic toolkit with new tools for precise cellular control.

Main Methods:

• Bioinformatic screening of sequence databases for novel ChRs.
• Electrophysiological recordings in mammalian cells.
• Spectroscopic measurements (transient absorption) and pH titrations.
• Site-directed mutagenesis and functional analysis.
• Optogenetic activation and silencing experiments in brain slices and live organisms.

Main Results:

• Identification of three naturally blue-shifted ancyromonad ChRs, forming a distinct phylogenetic group.
• One homologue, Nutomonas longa, generates potent cation currents at ~440 nm, surpassing existing tools.
• Characterization of key residues in the photoactive site influencing channel function.
• Demonstration of two-photon activation for deep-tissue optogenetics.
• Successful optogenetic silencing of mouse cortical neurons and photoinhibition of muscle contraction in worms.

Conclusions:

• Ancyromonad ChRs represent a new class of potent, blue-shifted optogenetic tools.
• These findings enhance the mechanistic understanding of light-gated channel operation.
• The discovered ChRs significantly expand the available optogenetic toolkit for neuroscience and beyond.