Enzymatic vitamin A2 production enables red-shifted optogenetics

Abstract

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.

Keywords: ChR2; Cyp27c1; Optogenetics; ReaChR; Vitamin A2.

Fig. 1

Spectral sensitivity of ChR2. a Expression of ChR2 in fusion to EYFP (yellow) in HEK 293. b Representative ChR2 currents evoked by illumination (480 nm, 0.5 s, grey line) in ChR2 expressing HEK 293 cells in Panserin 293A medium supplemented with 25 μM vitamin A₁ (A1) or vitamin A₂ (A2) 24-48 h before experiments. c ChR2 steady-state photocurrent at different wavelengths in relation to the maximal current for each cell in the presence of vitamin A₁ (25 μM, n = 15) or A₂ (25 μM, n = 17). Spectral difference at 50% photocurrent highlighted by black arrow. d Ratio of normalized ChR2 currents in the presence of vitamin A₂ and A₁. e,f Quantification of normalized (e) and absolute (f) ChR2 photocurrents at 550 nm in the presence of vitamin A₁ and A₂. **** p ≤ 0.0001

Fig. 2

Spectral sensitivity of ReaChR. a Expression of ReaChR in fusion to Cerulean (blue) in HEK 293 cells. b Representative ReaChR currents evoked by illumination (600 nm, 0.5 s, grey line) in ReaChR expressing HEK 293 cells in Panserin 293A medium supplemented with 25 μM vitamin A₁ (A1) or vitamin A₂ (A2) 24-48 h before experiments. c ReaChR steady-state photocurrent at different wavelengths in relation to the maximal current for each cell in the presence of vitamin A₁ (25 μM, n = 10) or A₂ (25 μM, n = 10). Spectral difference at 50% photocurrent highlighted by black arrow. d Ratio of normalized ReaChR currents in the presence of vitamin A₂ and A₁. e,f Quantification of normalized (e) and absolute (f) ReaChR photocurrents at 650 nm in the presence of vitamin A₁ and A₂. **** p ≤ 0.0001

Fig. 3

Cyp27c1 expression converts vitamin A₁ into vitamin A₂. a Plasmid for expression of zebrafish Cyp27c1 and EGFP separated by a 2A self-cleaving peptide. b,c HEK 293 cells stably expressing Cyp17c1-EGFP (green) in overview (b) and stained with antibodies (c) against Cyp27c1 (red) and mitochondrial ATP synthase F1-β (white). d HPLC analysis of HEK 293 cells without (CTR) and with Cyp27c1 (CYP) expression after incubation with 3.5 μM vitamin A₁ (all-trans retinol). Specific peaks for all-trans retinol (AT-ROL) and 13-cis retinol (13c-ROL) species of vitamin A₁ and A₂ highlighted in grey. Note the appearance of vitamin A₂ species only in Cyp27c1 expressing cells. e,f Quantification of total amount of vitamin A₁ (e, n = 4) or vitamin A₂ (f, n = 4) normalized to total protein level in HEK 293 cells without (CTR) and with Cyp27c1 (CYP) expression after incubation with vitamin A₁ (pooled data for 3.5 and 35 μM vitamin A₁). ** p ≤ 0.01

Fig. 4

Cyp27c1 co-expression red-shifts the spectral sensitivity of ChR2 a Transient expression of ChR2-EYFP (yellow) in a HEK 293 cell line stably expressing Cyp27c1-EGFP (green). Special fluorescence filters were chosen to discriminate between EYFP and EGFP. b ChR2 photocurrents in relation to the maximal current for each cell in Panserin 293A medium for cells expressing Cyp27c1 without (CYP, n = 14) and with vitamin A₁ incubation (CYP + A1, 5 μM, n = 11) and in control cells without (CTR, n = 19) and with vitamin A₂ incubation (A2, 25 μM, n = 17). Note that Cyp27c1 expression shifts the spectral sensitivity of ChR2 at 50% photocurrent (black arrow). c Ratio of normalized ChR2 photocurrents in cells expressing Cyp27c1 and control cells shows up to three times higher currents at wavelengths between 550 and 600 nm. d Wavelengths resulting in 50% of ChR2 photocurrents from data shown in b calculated for each cell by linear fitting. e,f Quantification of normalized (e) and absolute (f) ChR2 photocurrents at 550 nm.. * p ≤ 0.05, ** p ≤ 0.01, **** p ≤ 0.0001

Fig. 5

Cyp27c1 co-expression red-shifts spectral sensitivity of ReaChR a Transient expression of ReaChR-Cerulean (blue) in a HEK 293 cell line stably expressing Cyp27c1-EGFP (green). b ReaChR photocurrents in relation to the maximal current for each cell in Panserin 293A medium for cells expressing Cyp27c1 without (CYP, n = 15) and with vitamin A₁ incubation (CYP + A1, 5 μM, n = 14) and in control cells without (CTR, n = 15) and with vitamin A₂ incubation (A2, 25 μM, n = 14). Note that Cyp27c1 expression shifts the spectral sensitivity of ReaChR at 50% photocurrent (black arrow). c Ratio of normalized ReaChR photocurrents in cells expressing Cyp27c1 and control cells shows more than three times higher currents at wavelengths above 650 nm. d Wavelengths resulting in 50% of normalized ReaChR photocurrents from data shown in b calculated for each cell by linear fitting e,f Quantification of normalized (e) and absolute (f) ReaChR photocurrents at 650 nm from data shown in b. * p ≤ 0.05, ** p ≤ 0.01, **** p ≤ 0.0001