Abstract
Purpose:
Channelrhodopsons (ChRs), such ChR2, are promising optogenetic light sensors for vision restoration. A major obstacle for using ChRs in vision restoration is their low light sensitivity. Several ChR2 mutants have been reported to increase light sensitivity but with a trade-off in slower kinetics. Recently, a large number of ChR variants have been reported by de novo transcriptome sequencing of algae (Klapoetke et al., 2014). One of the variants, CoChR, displayed large photocurrents. Here, we investigated the light response properties of CoChR, especially its light sensitivity, and compared with the previously reported highly light-sensitive ChR2 mutants. We are also conducting molecular engineering aiming to further optimize its light sensitivity and kinetics for vision restoration.
Methods:
CoChR fused with GFP was cloned into a mammalian expression vectors with CAG promoter. Mutations were produced by site-directed mutagenesis. The expression and light response properties were evaluated in HEK cells. Light response properties were examined by whole-cell patch-clamp recordings.
Results:
CoChR exhibited robust expression and large photocurrents. Its peak spectrum showed a moderate red-shifted compared to ChR2. The photocurrents evoked by high light intensity displayed moderate desensitization. The off-rate of the photocurrents is ~200 ms, which is similar to that of ChR2(L132C/T159C), one of the previously reported highly light-sensitive ChR2 mutants. The photocurrents of CoChR evoked by low light intensities were 2 - 3 folds larger than that of ChR2(L132C/T159C). Furthermore, we created a number of CoChR mutants with altered photocurrent desensitization and off-rate. The photocurrents to low light intensities were further increased for some of these mutants with slower kinetics. Our ongoing studies are examining the light sensitivity and kinetics from CoChR-expressing retinal ganglion cells in mice by AAV vector-mediated delivery.
Conclusions:
CoChR exhibits higher light sensitivity compared to the ChR2 mutants of comparable kinetics. Its light sensitivity and temporal properties can be further optimized by molecular engineering. Thus, CoChR or its mutant(s) could be better optogenetic light sensors for vision restoration.