Opsins are light-sensitive G-protein-coupled receptors. They are mainly involved in vision and also have a few non-visual functions such as regulation of the circadian rhythm, pupillary light reflex, mood, behavior regulation, retina development, ect. Opsins utilize retinal as the chromophore. In the dark, opsins bind to 11-cis-retinal and converts to all-trans-retinal upon light exposure. Monostable opsins are thermally unstable in the all-trans-retinal-bound form, which leads to hydrolysis of the retinal. In contrast, bistable opsins are stable in both states, which allows them to toggle between 11-cis and all-trans retinal bound states in the presence of light. Till date, over 2,000 opsins have been identified, however, only a few have been characterized. Characterization of opsins allows for the realization of the different signaling abilities and spectral selectivity of opsins, which is highly beneficial in subcellular and in-vivo optogenetic applications. Monostable opsins require an exogenous supply of retinal to function outside the retina unlike bistable opsins. Since bistable opsins can recycle retinal, they are active even at low serum level concentrations of retinal. Therefore, identifying and characterization of bistable opsins with distinct signaling abilities are of considerable interest. This will allow for development of better optogenetic modules. Here, we propose an assay to detect the stability status of opsins. In this, we tested the signaling activity of a known bistable opsin, melanopsin, and a known monostable opsin, blue opsin, in a low level of 11-cis-retinal concentration (50 nM). We observed that the monostable opsin showed initial activation, however, completely deactivated within a few minutes. However, the bistable opsin stayed active. Photoconversion of the retinal bound to blue opsin (monostable) leads to the hydrolysis of the chromophore from the opsin, which in turn inactivates the receptor. However, since melanopsin (bistable) is stable at both states, it stays active. These results indicate the assay worked successfully and this can be extended to identify and characterize the stability status of other unknown opsins.

Detection of photoreceptor bistability and monostability: An assay

ACS Spring 2022

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