Chimeric human opsins as optogenetic light sensitizers

Abstract

Purpose : Human opsin-based photopigments have great potential as light-sensitizers, but their requirement for phototransduction cascade-specific second messenger proteins limits their functionality in non-native cell types. Human chimeric opsins where intracellular domains of representative visual opsins were replaced by corresponding regions of G_q/11-coupled melanopsin were investigated as potential optogenetic tools.

Methods : Eight chimeric human opsins were generated that consisted of either a human rod opsin or long-wavelength-sensitive opsin (LWS) backbone with intracellular domains that were replaced with corresponding regions derived from human melanopsin. Opsin protein trafficking and G_i and G_q/11 second messenger coupling were investigated by immunocytochemistry, and cAMP and calcium live-cell assays. Chromophore requirements were also investigated.

Results : Rhodopsin/melanopsin chimeric pigments were able to couple to both G_q/11 and G_i pathways to varying degrees: greater substitution of the intracellular surface with corresponding melanopsin domains generally showed greater G_q/11 activity with a decrease in the ability to activate G_i (area under the curve (AUC) analysis for each dataset: P < 0.0001, ordinary one-way ANOVA; n = 11 and 8, respectively). The relative time to peak G_q/11 fluorescence showed statistically significant differences between all groups (F (6, 62) = 6.4, P < 0.0001, ordinary one-way ANOVA; n = 11). Unlike melanopsin, rhodopsin and rhodopsin/melanopsin chimeric opsins were found to require exogenous 9-cis retinal to function. Wild type LWS opsin and LWS/melanopsin chimeras showed only minor G_i activation (F (6, 21) = 0.274, P = 0.142, ordinary one-way ANOVA; n = 4) and no G_q/11 activity in response to light stimulation. Immunocytochemistry demonstrated that chimeric opsins with more intracellular domains derived from melanopsin were less likely to be trafficked to the plasma membrane and more likely to lead to the formation of aggresomes.

Conclusions : By creating these unique human opsins that combine the spectral and chromophore requirements of rhodopsin with the intracellular signaling properties of melanopsin, this study demonstrates the importance of G_α coupling efficiency to the speed of cellular responses, and created human opsins with a unique combination of properties to expand the range of customised optogenetic biotools for basic research and translational therapies.

This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.