Animal opsins are light-sensitive G-protein-coupled receptors (GPCRs) that enable optogenetic control over the major heterotrimeric G-protein signaling pathways in animal cells. As such, opsins have potential applications in both biomedical research and therapy. Selecting the opsin with the best balance of activity and selectivity for a given application requires knowing their ability to couple to a full range of relevant Gα subunits. We present the GsX assay, a set of tools based on chimeric Gs subunits that transduce coupling of opsins to diverse G proteins into increases in cAMP levels,  measured with a real-time reporter in living cells. We use this assay to compare coupling to Gi/o/t across a panel of natural and chimeric opsins selected for potential application in gene therapy for retinal degeneration.

Photoreception by vertebrates enables both image-forming vision and non-image-forming responses such as circadian photoentrainment. Over the recent years, distinct non-rod non-cone photopigments have been found to support circadian photoreception in diverse species. By allowing specialization to this sensory task a selective advantage is implied, but the nature of that specialization remains elusive. We have used the presence of distinct rod opsin genes specialized to either image-forming (retinal rod opsin) or non-image-forming (pineal exo-rod opsin) photoreception in ray-finned fish (Actinopterygii) to gain a unique insight into this problem. A comparison of biochemical features for these paralogous opsins in two model teleosts, Fugu pufferfish (Takifugu rubripes) and zebrafish (Danio rerio), reveals striking differences. While spectral sensitivity is largely unaltered by specialization to the pineal environment, in other aspects exo-rod opsins exhibit a behavior that is quite distinct from the cardinal features of the rod opsin family. While they display a similar thermal stability, they show a greater than tenfold reduction in the lifetime of the signaling active Meta II photoproduct. We show that these features reflect structural changes in retinal association domains of helices 3 and 5 but, interestingly, not at either of the two residues known to define these characteristics in cone opsins. Our findings suggest that the requirements of non-image-forming photoreception have lead exo-rod opsin to adopt a characteristic that seemingly favors efficient bleach recovery but not at the expense of absolute sensitivity.

Introduction: Intrinsically photosensitive retinal ganglion cells (ipRGCs) integrate melanopsin and rod/cone-mediated inputs to signal to the brain. Whilst originally identified as a cell type specialised for encoding ambient illumination, several lines of evidence indicate a strong association between colour discrimination and ipRGC-driven responses. Thus, cone-mediated colour opponent responses have been widely found across ipRGC target regions in the mouse brain and influence a key ipRGC-dependent function, circadian photoentrainment. Although ipRGCs exhibiting spectrally opponent responses have also been identified, the prevalence of such properties have not been systematically evaluated across the mouse retina or yet been found in ipRGC subtypes known to influence the circadian system. Indeed, there is still uncertainty around the overall prevalence of cone-dependent colour opponency across the mouse retina, given the strong retinal gradient in S and M-cone opsin (co)-expression and overlapping spectral sensitivities of most mouse opsins. Methods: To address this, we use photoreceptor isolating stimuli in multielectrode recordings from human red cone opsin knock-in mouse (Opn1mwR) retinas to systematically survey cone mediated responses and the occurrence of colour opponency across ganglion cell layer (GCL) neurons and identify ipRGCs based on spectral comparisons and/or the persistence of light responses under synaptic blockade. Results: Despite detecting robust cone-mediated responses across the retina, we find cone opponency is rare, especially outside of the central retina (overall ~3% of GCL neurons). In keeping with previous suggestions we also see some evidence of rod-cone opponency (albeit even more rare under our experimental conditions), but find no evidence for any enrichment of cone (or rod) opponent responses among functionally identified ipRGCs. Conclusion: In summary, these data suggest the widespread appearance of cone-opponency across the mouse early visual system and ipRGC-related responses may be an emergent feature of central visual processing mechanisms.