Melanopsin1,2,3,4,5,6,7,8 has been proposed to be the photopigment of the intrinsically photosensitive retinal ganglion cells (ipRGCs)7,8,9,10,11,12,13,14,15; these photoreceptors of the mammalian eye drive circadian and pupillary adjustments through direct projections to the brain5,6,8,9,10,11,12,13,14,16,17,18. Their action spectrum (λmax ≈ 480 nm) implicates an opsin10 and melanopsin is the only opsin known to exist in these cells. Melanopsin is required for ipRGC photosensitivity13 and for behavioural photoresponses that survive disrupted rod and cone function14,17. Heterologously expressed melanopsin apparently binds retinaldehyde and mediates photic activation of G proteins19. However, its amino-acid sequence differs from vertebrate photosensory opsins1,20 and some have suggested that melanopsin may be a photoisomerase, providing retinoid chromophore to an unidentified opsin3,20. To determine whether melanopsin is a functional sensory photopigment, here we transiently expressed it in HEK293 cells that stably expressed TRPC3 channels. Light triggered a membrane depolarization in these cells and increased intracellular calcium. The light response resembled that of ipRGCs, with almost identical spectral sensitivity (λmax ≈ 479 nm). The phototransduction pathway included Gq or a related G protein, phospholipase C and TRPC3 channels. We conclude that mammalian melanopsin is a functional sensory photopigment, that it is the photopigment of ganglion-cell photoreceptors, and that these photoreceptors may use an invertebrate-like phototransduction cascade.
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We thank V. Maine for technical support; M. Zhu for donating the HEK293-TRPC3 cells; T. Helton, K. Richard, D. Lipscombe, J. Bai and X. Wang for guidance with cell culture; E. Newman and D. O'Malley for advice on calcium imaging; and M. Rollag and J. McIlwain for discussions and comments on the manuscript. This work was supported by NIH grants to D.M.B. and I.P.
The authors declare that they have no competing financial interests.
Evidence (1) that the light-activated current in melanopsin expressing HEK293-TRPC3 cells is carried by TRPC3 channels; and (2) that the signaling cascade shares components with the M1 muscarinic receptor cascade. It discusses effects on the light response of altering or eliminating supplemental retinoids. (DOC 32 kb)
Additional details on cell culture, expression of melanopsin, electrophysiological and pharmacological techniques, photic stimulation methods, spectral analysis, and calcium imaging. (DOC 38 kb)
Light evokes calcium responses in HEK293-TRPC3 cells that express melanopsin but not EGFP. (DOC 275 kb)
Evidence that (A) U73343, the inactive analog of the PLC blocker U73122, was without effect; (B) bath application of lanthanum abolishes the light response; and (C) inward currents evoked by the M1 muscarinic agonist carbachol occlude the light response in melanopsin-expressing HEK293-TRPC3 cells. (DOC 1602 kb)
Cumulative irradiance-response functions at the six wavelengths tested, pooled across all recorded cells. (DOC 124 kb)
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Qiu, X., Kumbalasiri, T., Carlson, S. et al. Induction of photosensitivity by heterologous expression of melanopsin. Nature 433, 745–749 (2005). https://doi.org/10.1038/nature03345
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