Enzymatic vitamin A₂ production enables red-shifted optogenetics

Optogenetics is a technology using light-sensitive proteins to control signaling pathways and physiological processes in cells and organs and has been applied in neuroscience, cardiovascular sciences, and many other research fields. Most commonly used optogenetic actuators are sensitive to blue and green light, but red-light activation would allow better tissue penetration and less phototoxicity. Cyp27c1 is a recently deorphanized cytochrome P450 enzyme that converts vitamin A₁ to vitamin A₂, thereby red-shifting the spectral sensitivity of visual pigments and enabling near-infrared vision in some aquatic species. Here, we investigated the ability of Cyp27c1-generated vitamin A₂ to induce a shift in spectral sensitivity of the light-gated ion channel Channelrhodopsin-2 (ChR2) and its red-shifted homolog ReaChR. We used patch clamp to measure photocurrents at specific wavelengths in HEK 293 cells expressing ChR2 or ReaChR. Vitamin A₂ incubation red-shifted the wavelength for half-maximal currents (λ_50%) by 6.8 nm for ChR2 and 12.4 nm for ReaChR. Overexpression of Cyp27c1 in HEK 293 cells showed mitochondrial localization, and HPLC analysis showed conversion of vitamin A₁ to vitamin A₂. Notably, the λ_50% of ChR2 photocurrents was red-shifted by 10.5 nm, and normalized photocurrents at 550 nm were about twofold larger with Cyp27c1 expression. Similarly, Cyp27c1 shifted the λ_50% of ReaChR photocurrents by 14.3 nm and increased normalized photocurrents at 650 nm almost threefold. Since vitamin A₂ incubation is not a realistic option for in vivo applications and expression of Cyp27c1 leads to a greater red-shift in spectral sensitivity, we propose co-expression of this enzyme as a novel strategy for red-shifted optogenetics.