Abstract
Purpose:
Channelrhodopsin-2 (ChR2) is one of the most promising tools for the optogenetic-based vision restoration. A major disadvantage of using ChR2 for vision restoration, however, is its low light sensitivity. Recently, two ChR2 mutants, L132C and T159C, were reported to show a marked increase in light sensitivity (Kleinlogel et al., 2011; Berndt et al., 2011; Prigge et al., 2012). In this study, we created a number of ChR2 mutants at these two sites and evaluated their light response properties and expression safety in retinal neurons for developing more light-sensitive ChR2 variants for vision restoration.
Methods:
A number of single and double ChR2 mutations at the L132 and T159 sites were made. Their expression and light response properties were first examined in HEK cells. AAV2 vectors carrying the ChR2-GFP driven by CAG promoter were produced and injected intravitreally into the eyes of adult C57BL/6J mice. ChR2-mediated light sensitivity and kinetics of retinal ganglion cells (RGCs) were examined by multi-electrode array recordings in retinal whole-mounts. The expression safety of ChR2-mutants in the retina was assessed by immunostaining and cell counting after exposure to strong blue light for two weeks.
Results:
Consistent with the previous reports, the ChR2 mutants, L132C and/or T159C, exhibited a marked increase in light sensitivity. Several new mutants, including L132A/T159C and L132C/T159S, were found to further increase the light sensitivity. The higher light sensitivity, however, was found to be correlated with slower channel kinetics. The higher light sensitivity, however, was correlated with slower channel kinetics. The light sensitivity of L132C/T159C and L132C/T159S was increased by ~1.5 and 2 log units comparing to the wild-type ChR2 whereas the off rate was increased to ~180 and ~930 ms, respectively. The spiking activity of RGCs expressing L132C/T159C and L132C/T159S could follow flickering stimuli up to 15 and 5 Hz, respectively. The expression of these ChR2 mutants was not found to cause neurotoxicity up to two months after viral injection.
Conclusions:
We created several highly light-sensitive ChR2 mutants. There is always a trade-off between light sensitivity and channel kinetics. The ChR2 mutants with a balance between light sensitivity and channel kinetics, such as L132C/T159C or L132C/T159S, may be suitable for the application of vision restoration.
Keywords: 533 gene/expression •
625 opsins •
531 ganglion cells