A Bistable UV-Sensitive Opsin from a Reef Building Coral Showing a Switchable and Tunable Regulation of Gs-Signaling by Different Wavelengths of Light

Abstract Cnidarians possess a large number of opsins in their genomes for their various photoreceptive functions. In particular, they uniquely possess Gs-coupled opsins that induce intracellular cAMP accumulation in a light-dependent manner. These Gs-coupled opsins, cnidopsins, are powerful optogenetic tools that manipulate cAMP-dependent cellular responses. In this study, we characterized a cnidopsin, named AtCnidop3a, from the coral Acropora tenuis as a Gs-coupled and UV-sensitive bistable pigment. This cnidopsin showed a large spectral shift upon activation from absorption maxima from 395 to 560 nm, and the resting and activated states are interconvertible by illumination with UV (or violet) and orange light. The activated state efficiently activates Gs proteins and elevates intracellular cAMP levels in mammalian cultured cells. To engineer an opsin mutant that can be turned on and off upon long-wavelength light illumination by utilizing the large spectral separation, negatively charged amino acids were introduced near the retinal Schiff base region. Among the tested opsin mutants, the Y1133.28E mutant is capable of being activated by green light unlike the wild-type while retaining the property of being inactivated by orange light like the wild-type, indicating successful conversion of the opsin to a visible-light-sensitive bistable pigment. The visible-light-induced cAMP regulation of the Y1133.28E mutant was enhanced by an additional L942.61G substitution. Our characterization and engineering of the cnidopsin revealed the functional diversity of cnidarian opsins and their potential utility as optogenetic tools regulating Gs-dependent physiological responses.