Characterization and engineering of a blue-sensitive, Gi/o-biased, and bistable ciliary opsin from a fan worm

Abstract

Ciliary opsins have been identified not only in vertebrates but also in invertebrates. An invertebrate ciliary opsin was recently identified in the fan worm Acromegalomma interruptum (formerly named Megalomma interrupta); however, its spectral and signaling characteristics are unknown. In the present study, we characterized the spectral properties and light-induced cellular signaling properties of the opsin (AcrInvC-opsin). AcrInvC-opsin showed an absorption maximum at 464 nm and upon blue-light absorption, the spectrum was red-shifted by approximately 50 nm. The two states are inter-convertible by illumination with blue and orange light. Blue light illumination of AcrInvC-opsin caused specific coupling with Gi, sustained Gi dissociation, decreased intracellular cAMP levels, and activation of GIRK channels. The cellular responses by the activated opsin were partially terminated by orange light illumination. These light-dependent responses indicate that the InvC-opsin is a typical bistable pigment wherein the resting and activated states can be inter-converted by visible light illumination. We also attempted to modulate the spectral and functional properties of AcrInvC-opsin using site-directed mutagenesis. Substitution of Ser-94 with Ala caused little spectral shift in the resting state but a further red-shift of [~]10 nm in the activated state, indicating that the absorption spectra of the two states were tuned differently. In contrast, the S94A substitution did not significantly affect the light-dependent signaling properties of AcrInvC-opsin. Because AcrInvC-opsin is a blue-sensitive, Gi/o-biased, and bistable pigment, it has the potential to serve as an optical control tool to specifically and reversibly regulate Gi/o-dependent signaling pathways by visible light.