Blue-shifted ancyromonad channelrhodopsins for multiplex optogenetics.

Area of Science:

• Optogenetics and Channelrhodopsins
• Molecular Biology and Biophysics
• Neuroscience and Electrophysiology

Background:

• Light-gated ion channels, specifically channelrhodopsins (ChRs), are crucial optogenetic tools for controlling neuronal and cardiomyocyte activity.
• Developing spectrally distinct ChRs for multiplexing is challenging due to engineering difficulties that can impair channel function.
• Natural discovery of novel ChRs is essential for expanding the optogenetic toolkit for advanced applications.

Purpose of the Study:

• To identify and characterize novel, naturally blue-shifted channelrhodopsins from protists for optogenetic applications.
• To investigate the functional and mechanistic properties of newly discovered ancyromonad channelrhodopsins, particularly their ionic selectivity and photoactivation.
• To evaluate the efficacy of these novel channels as tools for optogenetic control in complex biological systems.

Main Methods:

• Bioinformatic screening of sequence databases to identify potential channelrhodopsin candidates from ancyromonads.
• Phylogenetic analysis to establish evolutionary relationships and identify distinct clades.
• Heterologous expression in mammalian cells, electrophysiological recordings, transient absorption spectroscopy, pH titrations, and site-directed mutagenesis to characterize channel function and photoactive site residues.
• In vivo optogenetic experiments in mouse cortical neurons and live C. elegans.

Main Results:

• Identification of three novel, blue-shifted channelrhodopsins from ancyromonads, forming a distinct phylogenetic group with anion channelrhodopsin (ACR) motifs.
• Characterization of a specific ancyromonad ACR (AnsACR) exhibiting strong inward cation currents in mammalian cells, surpassing existing tools, with a peak absorption around 440 nm.
• Demonstration of AnsACR's utility for optogenetic silencing of mouse cortical neurons and photoinhibition of pharyngeal muscle contraction in live worms, including activation via two-photon near-infrared illumination.

Conclusions:

• Ancyromonad channelrhodopsins represent a valuable new class of optogenetic tools with unique spectral properties and enhanced functionality.
• AnsACR provides a potent means for optogenetic control, particularly in thicker tissues due to its two-photon activation capability.
• This study enhances the understanding of light-gated channel mechanisms and significantly expands the available toolkit for optogenetics and all-optical electrophysiology.