Melanopsin functions in intrinsically photosensitive retinal ganglion cells of mammals to regulate circadian clock and pupil constriction. The opsinamide AA92593 has been reported to specifically inhibit mouse and human melanopsin functions as a competitive antagonist against retinal; however, the molecular mechanisms underlying its specificity have not been resolved. In this study, we attempted to identify amino acid residues responsible for the susceptibility of mammalian melanopsins to AA92593. Our cell-based assays confirmed that AA92593 effectively inhibited the light-induced cellular responses of mammalian melanopsins, but not those of non-mammalian vertebrate and invertebrate melanopsins. These results suggest that amino acid residues specifically conserved among mammalian melanopsins are important for the antagonistic effect of AA92593, and we noticed Phe-94^2.61, Ser-188^ECL2, and Ser-269^6.52 as candidate residues. Substitutions of these residues reduced the antagonistic effect of AA92593. We conducted docking and molecular dynamics simulations based on the AlphaFold-predicted melanopsin structure. The simulations indicated that Phe-94^2.61, Ser-188^ECL2, and Ser-269^6.52 are located at the AA92593-binding site, and additionally identified Trp-189^ECL2 and Leu-207^5.42 interacting with the antagonist. Substitutions of Trp-189^ECL2 and Leu-207^5.42 affected the antagonistic effect of AA92593. Furthermore, substitutions of these amino acid residues converted the AA92593-insensitive non-mammalian melanopsins susceptible to the antagonist. Based on experiments and molecular simulations, five amino acid residues, at positions 94^2.61, 188^ECL2, 189^ECL2, 207^5.42, and 269^6.52, were found to be responsible for the specific susceptibility of mammalian melanopsins to AA92593.

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