Robust optogenetic inhibition with red-light-sensitive anion-conducting channelrhodopsins

Area of Science:

• Optogenetics
• Neuroscience
• Molecular Biology

Background:

• Channelrhodopsins (ChRs) are crucial tools for controlling neuronal activity using light.
• Existing ChRs have limitations in spectral sensitivity and kinetic properties for certain applications.

Purpose of the Study:

• To discover and characterize novel anion-conducting channelrhodopsins (ACRs) from diverse phylogenetic origins.
• To engineer ACRs for enhanced optogenetic silencing with improved spectral and kinetic characteristics.

Main Methods:

• Phylogenetic analysis of ChR families to identify novel ACR candidates.
• Biophysical characterization of identified ACRs, including spectral sensitivity and photocurrent kinetics.
• Engineering of ACRs through site-directed mutagenesis and assessment of modified properties.

Main Results:

• Identification and characterization of MsACR1 from *Mantoniella squamata*, sensitive to yellow-green light (λmax at 555 nm).
• Engineering of a red-shifted and accelerated ACR (raACR) via a single amino acid substitution, enhancing red-light sensitivity and kinetics.
• Demonstration of both MsACR1 and raACR as effective optical silencers in mouse neurons (in vitro and in vivo) at low light intensities.

Conclusions:

• Novel ACRs offer versatile tools for optical neuronal silencing.
• Engineered raACR provides superior temporal resolution and red-shifted activation for advanced optogenetic control.