Abstract Channelrhodopsins (ChRs) are light-gated ion channels and invaluable tools for optogenetic applications. Recent developments in multicolor optogenetics, in which different neurons are controlled by multiple colors of light simultaneously, have increased the demand for ChR mutants with more distant absorption wavelengths. Here we report the 2.9 Å-resolution cryo-electron microscopy structure of a ChR from Klebsormidium nitens (KnChR), which is one of the most blue-shifted ChRs. The structure elucidates the 6- s-cis configuration of the retinal chromophore, indicating its contribution to a distinctive blue shift in action spectra. The unique architecture of the C-terminal region reveals its role in the allosteric modulation of channel kinetics, enhancing our understanding of its functional dynamics. Based on the structure-guided design, we developed mutants with blue-shifted action spectra. Finally, we confirm that UV or deep-blue light can activate KnChR-transfected precultured neurons, expanding its utility in optogenetic applications. Our findings contribute valuable insights to advance optogenetic tools and enable refined capabilities in neuroscience experiments.

Cryo-EM structure of a blue-shifted channelrhodopsin fromKlebsormidium nitens