Functional characterization of four opsins and two G alpha subtypes co-expressed in the molluscan rhabdomeric photoreceptor

BMC Biology · 8 authors, 6 centres

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This study reveals that the eye photoreceptor of the terrestrial slug *Limax* co-expresses four different visual opsins (Gq-rhodopsin, xenopsin, Opn5A, and Opn5B) with distinct spectral sensitivities ranging from UV to green, and that all four opsins can activate Go signaling, while only Gq-rhodopsin activates Gq. This complex opsin co-expression and G protein coupling may underlie the shift in spectral sensitivity observed between dark- and light-adapted states, providing a simple mechanism for fine-tuning vision without neural networks.

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**Background:** Rhabdomeric photoreceptors in the terrestrial slug *Limax* are unique because they co-express multiple visual opsins from different groups: Gq-coupled rhodopsin, xenopsin, Opn5A, and retinochrome. However, the molecular properties (spectral sensitivity, G protein selectivity) of these opsins were unknown, hindering understanding of the functional advantage of this multiplicity. This study aimed to characterize the molecular properties of these opsins and their G protein partners to elucidate their role in photoreception.

**Methods:** The authors generated a specific antibody against Opn5B and used immunohistochemistry to confirm its co-localization with other opsins in the rhabdomere of Type-I photoreceptors. Expression of G protein alpha subunits (Gq and Go) was analyzed by immunohistochemistry and in situ hybridization. Spectral sensitivities of the opsins were determined using heterologous action spectroscopy (HAS) based on light-induced cAMP changes in HEK293S cells expressing chimeric (for Gq-rhodopsin and xenopsin) or wild-type (for Opn5A and Opn5B) opsins. Absorption spectra of purified recombinant xenopsin and Opn5A were also measured. G protein activation was assessed using a NanoBiT-G-protein dissociation assay to test Gq and Go activation by each opsin.

**Key Results:** Opn5B was identified as the fourth visual opsin co-expressed in the rhabdomere of Type-I photoreceptors, along with Gq-rhodopsin, xenopsin, and Opn5A. Both Gq and Go alpha subunits were expressed in the rhabdomere. Spectral sensitivity measurements revealed: Gq-rhodopsin λmax ~456 nm; xenopsin λmax ~475 nm; Opn5A λmax ~500 nm with significant UV sensitivity; Opn5B λmax ~470 nm with significant UV sensitivity. Spectroscopic analysis confirmed xenopsin as a bistable photopigment with λmax ~475 nm and Opn5A with λmax ~500 nm. The NanoBiT assay showed that only Gq-rhodopsin activated Gq, while all four opsins (Gq-rhodopsin, xenopsin, Opn5A, Opn5B) activated Go. Retinochrome did not activate Gq or Gi/o signaling. The authors used least-squares fitting of ERG data from a previous study with the four opsin nomograms, showing that the shift in spectral sensitivity between dark-adapted (peak ~480 nm) and light-adapted (peak ~420 nm) states could be explained by changes in the relative contributions of the four opsins (e.g., coefficients for dark-adapted: Gq-rhodopsin 1.5, xenopsin -0.69, Opn5A 1.3, Opn5B -0.62; light-adapted: Gq-rhodopsin 2.0, xenopsin -1.2, Opn5A 0.47, Opn5B -0.38).

**Clinical Implications:** This study provides fundamental insights into the molecular basis of vision in a mollusk, demonstrating an unprecedented co-expression of four opsins from different classes in a single photoreceptor. The finding that multiple opsins with distinct spectral sensitivities and G protein coupling selectivities can fine-tune spectral sensitivity without neural networks has implications for understanding the evolution of visual systems and may inspire novel optogenetic tools. The complex G protein signaling (Gq and Go) activated by these opsins could be exploited for precise control of cellular signaling pathways in optogenetic applications.

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other

SPECIALTY

ophthalmology

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ORIGINAL PAPER

PMC10729476

Functional characterization of four opsins and two G alpha subtypes co-expressed in the molluscan rhabdomeric photoreceptor

BMC Biology

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ABSTRACT

Background Rhabdomeric photoreceptors of eyes in the terrestrial slug Limax are the typical invertebrate-type but unique in that three visual opsins (Gq-coupled rhodopsin, xenopsin, Opn5A) and one retinochrome, all belonging to different groups, are co-expressed. However, molecular properties including spectral sensitivity and G protein selectivity of any of them are not determined, which prevents us from understanding an advantage of multiplicity of opsin properties in a single rhabdomeric photoreceptor. To gain insight into the functional role of the co-expression of multiple opsin species in a photoreceptor, we investigated the molecular properties of the visual opsins in the present study. Results First, we found that the fourth member of visual opsins, Opn5B, is also co-expressed in the rhabdomere of the photoreceptor together with previously identified three opsins. The photoreceptors were also demonstrated to express Gq and Go alpha subunits. We then determined the spectral sensitivity of the four visual opsins using biochemical and spectroscopic methods. Gq-coupled rhodopsin and xenopsin exhibit maximum sensitivity at ~ 456 and 475 nm, respectively, and Opn5A and Opn5B exhibit maximum sensitivity at ~ 500 and 470 nm, respectively, with significant UV sensitivity. Notably, in vitro experiments revealed that Go alpha was activated by all four visual opsins, in contrast to the specific activation of Gq alpha by Gq-coupled rhodopsin, suggesting that the eye photoreceptor of Limax uses complex G protein signaling pathways. Conclusions The eye photoreceptor in Limax expresses as many as four different visual opsin species belonging to three distinct classes. The combination of opsins with different spectral sensitivities and G protein selectivities may underlie physiological properties of the ocular photoreception, such as a shift in spectral sensitivity between dark- and light-adapted states. This may be allowed by adjustment of the relative contribution of the four opsins without neural networks, enabling a simple strategy for fine-tuning of vision. Supplementary Information The online version contains supplementary material available at 10.1186/s12915-023-01789-7.

CITATION EXPORT

Ryota Matsuo, Mitsumasa Koyanagi, Tomohiro Sugihara et al.. Functional characterization of four opsins and two G alpha subtypes co-expressed in the molluscan rhabdomeric photoreceptor. BMC Biology. (2023). https://doi.org/10.1186/s12915-023-01789-7

AUTHORS

8 · 6 CENTRES

Ryota Matsuo, Mitsumasa Koyanagi, Tomohiro Sugihara, Taishi Shirata, Takashi Nagata, Keiichi Inoue, Yuko Matsuo, Akihisa Terakita

1https://ror.org/00skwgg83grid.411574.20000 0000 9681 1887International College of Arts and Sciences, Fukuoka Women’s University, 1-1-1 Kasumigaoka, Higashi-Ku, Fukuoka, 813-8529 Japan
2https://ror.org/01hvx5h04Department of Biology, Graduate School of Science, Osaka Metropolitan University, 3-3-138 Sugimoto, Sumiyoshi-Ku, Osaka, 558-8585 Japan
3grid.518217.80000 0005 0893 4200Department of Biology and Geosciences, Graduate School of Science, Osaka City University, 3-3-138 Sugimoto, Sumiyoshi-Ku, Osaka, 558-8585 Japan
4https://ror.org/01hvx5h04The OMU Advanced Research Institute of Natural Science and Technology, Osaka Metropolitan University, 3-3-138 Sugimoto, Sumiyoshi-Ku, Osaka, 558-8585 Japan
5https://ror.org/057zh3y96grid.26999.3d0000 0001 2151 536XThe Institute for Solid State Physics, The University of Tokyo, Kashiwa, Japan
6https://ror.org/00097mb19grid.419082.60000 0001 2285 0987PRESTO, Japan Science and Technology Agency, Kawaguchi, Japan