Color-tuned channelrhodopsins for multiwavelength optogenetics

Area of Science:

• Neuroscience
• Molecular Biology
• Biophysics

Background:

• Channelrhodopsins are essential tools in optogenetics for controlling neuronal activity.
• Channelrhodopsin-2 (ChR2) is widely used for neuronal excitation with blue light.
• There is a need for channelrhodopsins with altered spectral properties and kinetics for advanced applications.

Purpose of the Study:

• To engineer novel, high-efficiency channelrhodopsins with tunable spectral and kinetic properties.
• To create a panel of channelrhodopsins for independent dual-color optogenetic stimulation.
• To improve upon existing channelrhodopsin tools for neuroscience research.

Main Methods:

• Construction of chimeric channelrhodopsins by combining Chlamydomonas channelrhodopsin-1 and Volvox channelrhodopsin-1.
• Molecular engineering and kinetic fine-tuning of channelrhodopsin variants.
• Assessment of expression, plasma membrane integration, and photocurrents in HEK cells.
• Characterization of absorption maxima and open-state lifetimes.

Main Results:

• Engineered channelrhodopsin variants exhibited superb expression and plasma membrane integration.
• Photocurrents were up to 3-fold larger compared to channelrhodopsin-2.
• Developed chimeric variants with absorption maxima from 526 to 545 nm, complementing ChR2 derivatives (461-492 nm).
• Achieved fine-tuning of open-state lifetimes ranging from 19 ms to 5 s.
• Demonstrated independent activation of two neural populations using green- (560 nm) and blue-absorbing (405 nm) variants.

Conclusions:

• A novel panel of high-efficiency, color-tuned channelrhodopsins was successfully engineered.
• These variants offer improved performance and expanded spectral and kinetic ranges for optogenetics.
• The developed toolkit enables precise dual-color stimulation of distinct neural populations, advancing neuroscience research.