Development of visible light‐sensitive human neuropsin ( <scp>OPN5</scp> ) via single amino acid substitution

Neuropsin (Opn5), a UV-sensitive 'non-visual' opsin, has the potential to be used as optogenetic tools applicable to tissues outside of the eye because of its broad expression. However, its sensitivity to poorly tissue-penetrating UV light poses challenges for its application. In this study, we focused on human OPN5 (hOPN5) to identify amino acid(s) responsible for the UV sensitivity. Sequence alignment across UV-sensitive Opn5s identified a conserved lysine residue (Lys91) at a position implicated in spectral tuning in invertebrate opsins. Substitution of this residue with neutral or acidic amino acids caused substantial shifts in spectral sensitivity towards visible wavelengths. Our findings identify Lys91 as a key spectral tuning site in hOPN5 and provide visible-light-sensitive versions as a candidate for optogenetic applications. Impact statement A "non-visual" opsin, Opn5, is the only UV-sensitive opsin in human. In this study, we identified, for the first time, a key tuning site responsible for the UV sensitivity of hOPN5. In addition to its impact on the opsin molecular studies, this finding could pave the way for the development of novel visual-sensitive Opn5-based optogenetic tools.