ChR2 Mutants at L132 and T159 with Improved Operational Light Sensitivity for Vision Restoration

Abstract

The ectopic expression of microbial opsin-based optogenetic sensors, such as channelrhodopsin-2 (ChR2) in surviving inner retinal neurons, is a promising approach to restoring vision after retinal degeneration. However, a major limitation in using native ChR2 as a light sensor for vision restoration is the low light sensitivity of its expressing cells. Recently, two ChR2 mutations, T159C and L132C, were reported to produce higher photocurrents or have ultra light sensitivity. In this study, we created additional ChR2 mutants at these two sites to search for more light responsive ChR2 forms and evaluate their suitability for vision restoration by examining their light responsive properties in HEK cells and mouse retinal ganglion cells. We found additional ChR2 mutants at these two sites that showed a further increase in current amplitude at low light levels in the cells expressing these mutants, or operational light sensitivity. However, the increase in the operational light sensitivity was correlated with a decrease in temporal kinetics. Therefore, there is a trade-off between operational light sensitivity and temporal resolution for these more light responsive ChR2 mutants. Our results showed that for the two most light responsive mutants, L132C/T159C and L132C/T159S, the required light intensities for generating the threshold spiking activity in retinal ganglion cells were 1.5 and nearly 2 log units lower than wild-type ChR2 (wt-ChR2), respectively. Additionally, their ChR2-mediated spiking activities could follow flicker frequencies up to 20 and 10 Hz, respectively, at light intensities up to 1.5 log units above their threshold levels. Thus, the use of these more light responsive ChR2 mutants could make the optogenetic approach to restoring vision more feasible.

Optogenetic Approaches to Restoring Vision

“…Basically, a ChR with high light sensitivity requires production of a large photocurrent at low light stimulation, which is also defined as operational light sensitivity (referred to as light sensitivity for simplicity hereafter) (Mattis et al 2012). On the basis of this criterion, the L132C/T159S ChR2 mutant exhibits highest light sensitivity (Pan et al 2014a). A side-byside comparison of almost all the reported ChR variants revealed that the increase in the light sensitivity closely correlated with the slower kinetics or the increase in off-rate (Mattis et al 2012, Pan et al 2014a; this suggests that the increase in light sensitivity was mainly due to the prolonged channel opening, which decreases the temporal resolution.…”

“…On the basis of this criterion, the L132C/T159S ChR2 mutant exhibits highest light sensitivity (Pan et al 2014a). A side-byside comparison of almost all the reported ChR variants revealed that the increase in the light sensitivity closely correlated with the slower kinetics or the increase in off-rate (Mattis et al 2012, Pan et al 2014a; this suggests that the increase in light sensitivity was mainly due to the prolonged channel opening, which decreases the temporal resolution. When expressed in retinal ganglion cells, the respective light sensitivities of the L132C/T159C and L132C/T159S ChR2 mutants were 1.5 and nearly 2 log units higher than in wild-type ChR2 (Figure 3), whereas the modulation of their spike activities in response to low light followed light flicker frequencies up to 20 and 10 Hz, respectively (Pan et al 2014a).…”

Biophysics of Channelrhodopsin

“…Moreover, properties that strongly depend on the target cells, such as expression level and membrane targeting, are of serious and equal importance, and these should be individually tested for each ChR variant and cell type, respectively. Finally, to help users choose a ChR variant (30,39) CatCh (ChR2 L132C) Enhanced Mg 2+ and Ca 2+ selectivity, large photocurrents, low inactivation (34,57) ChR2 T159C Large photocurrents, improved retinal binding affinity (7,74) CatCh + (ChR2 L132C-T159C) Enhanced Mg 2+ and Ca 2+ selectivity, large photocurrents, low inactivation, provides high light sensitivity to host cells, more stable expression than CatCh (52,57,63) ChETA (ChR2 E123T-T159C) Fast photocycle at the expense of reduced transported charge per absorbed photon, reduced voltage dependency of channel closing kinetics (7,21) Chronos Very fast photocycle, large photocurrents, inverse voltage dependency of off kinetics, provides high light sensitivity to host cells (33) Step-function rhodopsins …”

A New Promoter Allows Optogenetic Vision Restoration with Enhanced Sensitivity in Macaque Retina

“…6 As an alternative to retinal prosthesis, optogenetics can be used to restore vision by expressing optical neuromodulators such as channelrhodopsins or photochemically modified mammalian ion channels in residual retinal neurons. [7][8][9][10][11][12][13][14][15] Expression of channelrhodopsin in RGCs might allow greater spatial resolution and acuity than that achieved with current prosthetic devices. This has motivated a large body of proof-of-concept studies in rodents and other small animals, which have shown the feasibility of the approach, [7][8][9][10][16][17][18] and a first-in-human phase 1 clinical trial was initiated recently (ClinicalTrials.gov NCT02556736).…”

“…Thus far, studies assessing the feasibility of using microbial opsins for vision restoration have been carried out in small laboratory animals such as rodents, rabbits, and marmosets. [7][8][9][10]26,27 To our knowledge, the viral doses and construct optimizations necessary to obtain functional optogenetic readout have not yet been investigated in macaques.…”