Self-regenerating opsin from reef-building coral

Abstract

Opsins underlie diverse physiological responses to light in animals. In the dark, most opsins bind the chromophore 11-cis retinal, which isomerizes to all-trans form upon light absorption, representing the initial key step in signaling. Maintenance of opsin function therefore requires continuous regeneration of the inactive, 11-cis-retinal-bound state. Here, we report a novel type of opsin, AtAntho2c, from a reef-building coral, whose active form, bound to all-trans retinal, can thermally revert to the initial dark state bound to 11-cis retinal. A cysteine residue in extracellular loop 2 region plays a key role in the self-regeneration ability. Using time-resolved and low-temperature spectroscopies, we identify two spectrally distinct photointermediates prior to the all-trans to 11-cis isomerization in AtAntho2c, whose formation rates and yields are found to vary depending on temperature and pH conditions. The active form of AtAntho2c activates Gi/o G protein, resulting in a transient and repeatable decrease in cellular cAMP levels upon repeated light stimulations, even in the absence of exogenous retinal in cultured cells. Furthermore, we confirm that cells expressing AtAntho2c exhibit membrane hyperpolarization via GIRK channel activation light-dependently. These properties highlight the potential of AtAntho2c as a versatile optogenetic actuator capable of repeatedly modulate Gi/o signaling without retinal supplementation. Significance StatementLight-sensitive proteins, opsins, form active states upon light absorption, leading to intracellular G protein signaling and various cellular outputs. The active states require specific enzymatic machinery or another photon absorption to regenerate inactive opsins ready to respond to repeated light stimuli and maintain continuous responsiveness. In our study, we identify and analyze a coral opsin of which the active state rapidly and autonomously reverts to the inactive state in the dark through thermal isomerization of the retinal chromophore within the opsin. This regeneration mechanism allows the opsin to respond to repeated light stimuli at high temporal resolution and maintain large signal amplitude without the need for exogenous retinal making this opsin potentially useful for developing versatile optogenetic tools.