RubyACRs, nonalgal anion channelrhodopsins with highly red-shifted absorption

Area of Science:

• Microbiology
• Biophysics
• Optogenetics

Background:

• Channelrhodopsins are integral membrane proteins that function as light-gated ion channels.
• They are widely utilized in optogenetics to control neuronal activity with light.
• Anion channelrhodopsins (ACRs) are a subclass of channelrhodopsins that conduct anions.

Purpose of the Study:

• To discover and characterize novel anion channelrhodopsins (ACRs) with unique spectral properties.
• To investigate the potential of these ACRs for optogenetic applications.
• To explore the evolutionary diversity of ACRs in different microbial lineages.

Main Methods:

• Bioinformatic identification of novel ACRs from labyrinthulea and haptophyte algae.
• Heterologous expression and functional characterization of ACRs in a host system.
• Spectroscopic analysis to determine absorption maxima and spectral tuning.
• Electrophysiological recordings to analyze photocurrents and channel gating mechanisms.
• Site-directed mutagenesis to identify residues critical for spectral tuning.

Main Results:

• Discovery of two new ACR families from labyrinthulea and haptophyte algae.
• Identification of four labyrinthulea ACRs (RubyACRs) with red-shifted absorption maxima (590-610 nm), the most red-shifted known.
• Identification of three spectral tuning residues critical for the red-shifted absorption.
• Functional characterization of AlACR1 revealed biphasic photocurrent decay with weak voltage dependence, suggesting distinct gating mechanisms compared to cryptophyte ACRs.
• Discovery of blue, green, and red-absorbing ACRs within the labyrinthulea family, enabling color-sensitive photosensing.
• Demonstration of functional energy transfer from a fluorescent protein to the RubyACR chromophore.

Conclusions:

• The novel RubyACRs represent a significant expansion of the optogenetic toolkit, offering red-shifted light sensitivities for precise neuronal control.
• The identified spectral tuning residues provide insights into the molecular mechanisms underlying rhodopsin spectral tuning.
• The diverse spectral profiles of ACRs within labyrinthulea suggest sophisticated light-sensing capabilities.
• These findings highlight the untapped potential of microbial rhodopsins for biotechnological applications.