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, 292 (31), 12971-12980

A Ciliary Opsin in the Brain of a Marine Annelid Zooplankton Is Ultraviolet-Sensitive, and the Sensitivity Is Tuned by a Single Amino Acid Residue

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A Ciliary Opsin in the Brain of a Marine Annelid Zooplankton Is Ultraviolet-Sensitive, and the Sensitivity Is Tuned by a Single Amino Acid Residue

Hisao Tsukamoto et al. J Biol Chem.

Abstract

Ciliary opsins were classically thought to function only in vertebrates for vision, but they have also been identified recently in invertebrates for non-visual photoreception. Larvae of the annelid Platynereis dumerilii are used as a zooplankton model, and this zooplankton species possesses a "vertebrate-type" ciliary opsin (named c-opsin) in the brain. Platynereis c-opsin is suggested to relay light signals for melatonin production and circadian behaviors. Thus, the spectral and biochemical characteristics of this c-opsin would be directly related to non-visual photoreception in this zooplankton model. Here we demonstrate that the c-opsin can sense UV to activate intracellular signaling cascades and that it can directly bind exogenous all-trans-retinal. These results suggest that this c-opsin regulates circadian signaling in a UV-dependent manner and that it does not require a supply of 11-cis-retinal for photoreception. Avoidance of damaging UV irradiation is a major cause of large-scale daily zooplankton movement, and the observed capability of the c-opsin to transmit UV signals and bind all-trans-retinal is ideally suited for sensing UV radiation in the brain, which presumably lacks enzymes producing 11-cis-retinal. Mutagenesis analyses indicated that a unique amino acid residue (Lys-94) is responsible for c-opsin-mediated UV sensing in the Platynereis brain. We therefore propose that acquisition of the lysine residue in the c-opsin would be a critical event in the evolution of Platynereis to enable detection of ambient UV light. In summary, our findings indicate that the c-opsin possesses spectral and biochemical properties suitable for UV sensing by the zooplankton model.

Keywords: G protein-coupled receptor (GPCR); brain; molecular evolution; photobiology; rhodopsin; ultraviolet-visible spectroscopy (UV-Vis spectroscopy).

Conflict of interest statement

The authors declare that they have no conflicts of interest with the contents of this article

Figures

Figure 1.
Figure 1.
Characterization of Platynereis c-opsin. A, molecular phylogenetic relationship between Platynereis c-opsin and other ciliary and rhabdomeric opsins. The phylogenetic tree was constructed by the neighbor-joining method using MEGA6 software (76). The groups and subgroups in the opsin family are indicated. UV-sensitive opsins are highlighted in boldface. The bootstrap probabilities are indicated at each branch node, and a scale bar (0.2 substitutions per site) is also shown. B, absorption spectra of purified Platynereis c-opsin. Spectra in the dark (black), after UV illumination (red), and after subsequent yellow light (>480 nm) illumination (blue) are shown. The black and blue spectra are almost superimposed. In the dark, the λmax value is 383 nm in the UV-A region. Inset, absorption spectrum of Platynereis c-opsin in the dark in a wider wavelength range. C, HPLC analyses of the retinal configurations in purified Platynereis c-opsin. Chromatographs of the opsin in the dark (black), after UV light illumination (red), and after subsequent yellow light (>480 nm) illumination (blue) are shown. Peaks labeled 11 and AT indicate the 11-cis and all-trans isomers, respectively. Syn and Anti indicate respective isomers of the retinal oxime. D, photoresponses of a Xenopus oocyte expressing Platynereis c-opsin WT as well as GIRK1/GIRK2. Representative voltage clamp current recording data of an oocyte expressing Platynereis c-opsin with GIRK1/GIRK2 in the dark (black), after UV light illumination (red), and after subsequent yellow light (>480 nm) illumination (blue) are shown. Clamped voltage values are schematically indicated (see “Experimental Procedures”). E, Changes in the inward current upon light illumination. Current amplitudes recorded from the oocyte at −100 mV (red) and 40 mV (blue) as a function of time are shown. E–G, UV (violet bar) and yellow (>480 nm, orange bar) light illumination are indicated, and the black bar indicates that the oocyte was kept in the dark. The UV-dependent increase in inward current at −100 mV and little change at 40 mV indicate that the inward current through an inwardly rectifying potassium channel, GIRK1/GIRK2, was successfully measured. F, photoresponses of an oocyte injected with only GIRK1/GIRK2 (without c-opsin). Current amplitude at −100 mV recorded from the oocyte as a function of time is shown. The oocyte was incubated with 11-cis-retinal before measurement and illuminated with yellow (>480 nm) or UV light. G, decay of UV-induced current in light-dependent and light-independent manners. Current amplitude at −100 mV from a Xenopus oocyte expressing Platynereis c-opsin is plotted. Note that, after termination of UV illumination, the current was gradually reduced, and illumination with yellow light accelerated the current decay.
Figure 2.
Figure 2.
Ability of Platynereis c-opsin to bind exogenous all-trans-retinal directly. A, absorption spectra of purified WT Platynereis c-opsin that were incubated with 11-cis (black), all-trans (red), 13-cis (green), or 9-cis (blue) retinal. B, HPLC analyses determining retinal isomers actually bound to the opsin proteins. Chromatographs of samples after incubation with 11-cis (black), all-trans (red), 13-cis (green), or 9-cis (blue) retinal are shown. Peaks labeled 11, AT, and 9 indicate the 11-cis, all-trans, and 9-cis isomers, respectively. Syn and Anti indicate respective isomers of the retinal oxime. Note that incubation with 13-cis-retinal resulted in all-trans-retinal binding to the opsin proteins (green).
Figure 3.
Figure 3.
Lys-94 is essential for UV (UV-A) reception and transmission by Platynereis c-opsin. A, amino acid sequence alignment of opsins around position 94 in the second transmembrane helix. Position 94 is highlighted in boldface, and positions 86, 89, and 90 are also indicated. Lys-94 in Platynereis c-opsin is highlighted in red. UV-sensitive opsins are labeled UV. Lys-90 in Drosophila Rh3 can be regarded as Lys-89 because of a gap at the site. B, arrangement of 11-cis-retinal and position 94 in the second transmembrane helix. 11-cis-retinal (yellow) and positions 94 as well as 86, 89, and 90 (red spheres) in the crystal structure of bovine rhodopsin (Protein Data Bank code 1U19) (48) are shown. Lys-296, which forms the retinal Schiff base linkage, is also shown. C–J, absorption spectra of the Platynereis c-opsin mutants K94T (C), K94A (D), K94S (E), K94V (F), K94D (G), K94E (H), K94H (I), and K94R (J). The λmax values of the mutants are indicated. K–M, spectral changes of Platynereis c-opsin mutants K94T, K94A, and K94S upon light illumination. The absorption spectra of the Platynereis c-opsin mutants K94T (K), K94A (L), and K94S (M) in the dark (black), after illumination with blue (440 nm) light (red), and after subsequent illumination with orange-red (>580 nm) light (blue). N and O, photo-induced changes of current amplitudes at −100 mV from Xenopus oocytes expressing Platynereis c-opsin WT (N) or the K94T mutant (O). Illumination with UV (violet bar) and yellow light (>480 nm, orange bar) is indicated, and the black bar indicates that the oocytes were kept in the dark. P, comparison of photo-induced currents by Platynereis c-opsin WT and the K94T mutant. Differences in current amplitudes at −100 mV before and after light illumination are plotted. The error bars represent S.D. values (n = 9 for WT and n = 5 for K94T). Q, photo-induced changes of current amplitude at −100 mV from a Xenopus oocyte expressing the Platynereis c-opsin K94T mutant. The oocyte is illuminated by yellow (>480 nm) light followed by UV light.
Figure 4.
Figure 4.
Loss of binding ability for exogenous all-trans-retinal in the Platynereis c-opsin K94T mutant. A–D, absorption spectra of Platynereis c-opsin K94T mutant samples that were purified after incubation with 11-cis (A), all-trans (B), 13-cis (C), or 9-cis (D) retinal. The λmax values after incubation with 11-cis or 9-cis retinal are indicated. Note that, after incubation with all-trans or 13-cis retinal, no absorbance except protein absorbance at ∼280 nm was observed.

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