Development of visible light-sensitive human OPN5 via single amino acid substitution

Animal opsins, light-sensitive G-protein-coupled receptors (GPCRs), serve as primary light receptors in animals, supporting vision and providing light-dependent regulation of physiology and behaviour. Animal opsins also show great potential to be used as optogenetic tools that regulate cellular G protein signalling with light. Opn5 is a UV-sensitive "non-visual" opsin that is expressed in a wide range of tissues throughout body. Although Opn5 could be a good template for the development of optogenetic tools applicable to tissues outside of the eye because of its broad expression, its sensitivity to poorly tissue-penetrating UV light poses challenges for its application. In this study, we focused on human OPN5 (hOPN5) to attempt to identify amino acid(s) responsible for the UV sensitivity and to shift the spectral sensitivity to visible light. Sequence alignment across UV-sensitive Opn5s identified a conserved lysine reside (Lys91) at a position implicated in spectral tuning in invertebrate opsins. We applied site-directed mutagenesis to replace this residue with neutral (alanine) or acidic (glutamate or aspartate) amino acids. Heterologous action spectroscopy of these mutants revealed substantial shifts in spectral sensitivity (55-63nm) toward visible wavelengths. Our findings identify Lys91 as a key spectral tuning site in hOPN5 and provide visible-light sensitive versions of this protein as a candidate for optogenetic applications.