Abstract
ABSTRACTHuman opsin-based photopigments have great potential as light-sensitisers, but their requirement for phototransduction cascade-specific second messenger proteins may restrict their functionality in non-native cell types. In this study, eight chimeric human opsins were generated consisting of a backbone of either a rhodopsin (RHO) or long-wavelength-sensitive (LWS) opsin and intracellular domains from Gq/11-coupled human melanopsin. Rhodopsin/melanopsin chimeric opsins coupled to both Gi and Gq/11 pathways. Greater substitution of the intracellular surface with corresponding melanopsin domains generally showed greater Gq/11 activity with a decrease in Gi activation. Unlike melanopsin, rhodopsin and rhodopsin/melanopsin chimeras were dependent upon exogenous chromophore to function. By contrast, wild-type LWS opsin and LWS opsin/melanopsin chimeras showed only weak Gi activation in response to light, whilst Gq/11 pathway activation was not detected. Immunocytochemistry (ICC) demonstrated that chimeric opsins with more intracellular domains of melanopsin were less likely to be trafficked to the plasma membrane. This study demonstrates the importance of Gα 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.
Highlights
Optogenetics is the use of light-sensitive molecules to confer photosensitivity to cells that are not intrinsically photoreceptive, enabling the manipulation of cellular function by a light stimulus (Yizhar et al, 2011)
Immunolabelling with a rabbit polyclonal antibody raised against a peptide sequence corresponding to the distal C terminus of bovine rhodopsin showed strong labelling at the plasma membrane for cells transfected with RHO and RHO/OPN4 ICL3 (Fig. S3)
Rhodopsin/melanopsin chimeras can couple to two distinct G protein pathways This study has demonstrated that chimeras consisting of rhodopsin extracellular and transmembrane regions with melanopsin intracellular domains require exogenous chromophore and signal via both the Gq/11 and Gi pathways
Summary
Optogenetics is the use of light-sensitive molecules to confer photosensitivity to cells that are not intrinsically photoreceptive, enabling the manipulation of cellular function by a light stimulus (Yizhar et al, 2011). Several photosensitive molecules have been investigated as optogenetic tools for vision restoration, including human rhodopsin, melanopsin and medium wavelength opsin Received 26 November 2020; Accepted 8 June 2021 van Wyk et al, 2015; Gaub et al, 2015; Cehajic-Kapetanovic et al., 2015; Berry et al, 2019) These studies have shown some success, the light sensitivity and second messenger coupling of these photosensitive molecules are limited. There are clear advantages of coupling to a ubiquitous signalling pathway to facilitate amplification and increase light sensitivity
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