Induction of photosensitivity by heterologous expression of melanopsin

Abstract

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.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Figure 1: Light-evoked responses of melanopsin-expressing HEK293-TRPC3 cells.
Figure 4: Spectral sensitivity.
Figure 2: Light evokes calcium responses.
Figure 3: Phototransduction cascade.

References

  1. 1

    Provencio, I., Jiang, G., De Grip, W. J., Hayes, W. P. & Rollag, M. D. Melanopsin: An opsin in melanophores, brain, and eye. Proc. Natl Acad. Sci. USA 95, 340–345 (1998)

    CAS  Article  ADS  Google Scholar 

  2. 2

    Provencio, I. et al. A novel human opsin in the inner retina. J. Neurosci. 20, 600–605 (2000)

    CAS  Article  Google Scholar 

  3. 3

    Bellingham, J., Whitmore, D., Philp, A. R., Wells, D. J. & Foster, R. G. Zebrafish melanopsin: isolation, tissue localisation and phylogenetic position. Brain Res. Mol. Brain Res. 107, 128–136 (2002)

    CAS  Article  Google Scholar 

  4. 4

    Hannibal, J. & Fahrenkrug, J. Melanopsin: a novel photopigment involved in the photoentrainment of the brain's biological clock? Ann. Med. 34, 401–407 (2002)

    CAS  Article  Google Scholar 

  5. 5

    Hannibal, J., Hindersson, P., Knudsen, S. M., Georg, B. & Fahrenkrug, J. The photopigment melanopsin is exclusively present in pituitary adenylate cyclase-activating polypeptide-containing retinal ganglion cells of the retinohypothalamic tract. J. Neurosci. 22 RC191 (2002)

  6. 6

    Hattar, S., Liao, H. W., Takao, M., Berson, D. M. & Yau, K. W. Melanopsin-containing retinal ganglion cells: architecture, projections, and intrinsic photosensitivity. Science 295, 1065–1070 (2002)

    CAS  Article  ADS  Google Scholar 

  7. 7

    Provencio, I., Rollag, M. D. & Castrucci, A. M. Photoreceptive net in the mammalian retina. Nature 415, 493 (2002)

    CAS  Article  ADS  Google Scholar 

  8. 8

    Berson, D. M. Strange vision: ganglion cells as circadian photoreceptors. Trends Neurosci. 26, 314–320 (2003)

    CAS  Article  Google Scholar 

  9. 9

    Gooley, J. J., Lu, J., Chou, T. C., Scammell, T. E. & Saper, C. B. Melanopsin in cells of origin of the retinohypothalamic tract. Nature Neurosci. 4, 1165 (2001)

    CAS  Article  Google Scholar 

  10. 10

    Berson, D. M., Dunn, F. A. & Takao, M. Phototransduction by retinal ganglion cells that set the circadian clock. Science 295, 1070–1073 (2002)

    CAS  Article  ADS  Google Scholar 

  11. 11

    Panda, S. et al. Melanopsin (Opn4) requirement for normal light-induced circadian phase shifting. Science 298, 2213–2216 (2002)

    CAS  Article  ADS  Google Scholar 

  12. 12

    Ruby, N. F. et al. Role of melanopsin in circadian responses to light. Science 298, 2211–2213 (2002)

    CAS  Article  ADS  Google Scholar 

  13. 13

    Lucas, R. J. et al. Diminished pupillary light reflex at high irradiances in melanopsin-knockout mice. Science 299, 245–247 (2003)

    CAS  Article  ADS  Google Scholar 

  14. 14

    Panda, S. et al. Melanopsin is required for non-image-forming photic responses in blind mice. Science 301, 525–527 (2003)

    CAS  Article  ADS  Google Scholar 

  15. 15

    Warren, E. J., Allen, C. N., Brown, R. L. & Robinson, D. W. Intrinsic light responses of retinal ganglion cells projecting to the circadian system. Eur. J. Neurosci. 17, 1727–1735 (2003)

    Article  Google Scholar 

  16. 16

    Lucas, R. J., Douglas, R. H. & Foster, R. G. Characterization of an ocular photopigment capable of driving pupillary constriction in mice. Nature Neurosci. 4, 621–626 (2001)

    CAS  Article  Google Scholar 

  17. 17

    Hattar, S. et al. Melanopsin and rod-cone photoreceptive systems account for all major accessory visual functions in mice. Nature 424, 76–81 (2003)

    CAS  Article  ADS  Google Scholar 

  18. 18

    Gooley, J. J., Lu, J., Fischer, D. & Saper, C. B. A broad role for melanopsin in nonvisual photoreception. J. Neurosci. 23, 7093–7106 (2003)

    CAS  Article  Google Scholar 

  19. 19

    Newman, L. A., Walker, M. T., Brown, R. L., Cronin, T. W. & Robinson, P. R. Melanopsin forms a functional short-wavelength photopigment. Biochemistry 42, 12734–12738 (2003)

    CAS  Article  Google Scholar 

  20. 20

    Bellingham, J. & Foster, R. G. Opsins and mammalian photoentrainment. Cell Tissue Res. 309, 57–71 (2002)

    CAS  Article  Google Scholar 

  21. 21

    Hurst, R. S., Zhu, X., Boulay, G., Birnbaumer, L. & Stefani, E. Ionic currents underlying HTRP3 mediated agonist-dependent Ca2+ influx in stably transfected HEK293 cells. FEBS Lett. 422, 333–338 (1998)

    CAS  Article  Google Scholar 

  22. 22

    Belenky, M. A., Smeraski, C. A., Provencio, I., Sollars, P. J. & Pickard, G. E. Melanopsin retinal ganglion cells receive bipolar and amacrine cell synapses. J. Comp. Neurol. 460, 380–393 (2003)

    Article  Google Scholar 

  23. 23

    Fields, T. A. & Casey, P. J. Signalling functions and biochemical properties of pertussis toxin-resistant G-proteins. Biochem. J. 321, 561–571 (1997)

    CAS  Article  Google Scholar 

  24. 24

    Lamb, T. D. Photoreceptor spectral sensitivities: common shape in the long- wavelength region. Vision Res. 35, 3083–3091 (1995)

    CAS  Article  Google Scholar 

  25. 25

    Rollag, M. D., Provencio, I., Sugden, D. & Green, C. B. Cultured amphibian melanophores: a model system to study melanopsin photobiology. Methods Enzymol. 316, 291–309 (2000)

    CAS  Article  Google Scholar 

  26. 26

    Yoshimura, T. & Ebihara, S. Spectral sensitivity of photoreceptors mediating phase-shifts of circadian rhythms in retinally degenerate CBA/J (rd/rd) and normal CBA/N (+ / + )mice. J. Comp. Physiol. 178, 797–802 (1996)

    CAS  Article  Google Scholar 

  27. 27

    Brueggemann, L. I. & Sullivan, J. M. HEK293S cells have functional retinoid processing machinery. J. Gen. Physiol. 119, 593–612 (2002)

    CAS  Article  Google Scholar 

  28. 28

    Wong, S. K. G protein selectivity is regulated by multiple intracellular regions of GPCRs. Neurosignals 12, 1–12 (2003)

    CAS  Article  Google Scholar 

  29. 29

    Oh, E. J., Gover, T. D., Cordoba-Rodriguez, R. & Weinreich, D. Substance P evokes cation currents through TRP channels in HEK293 cells. J. Neurophysiol. 90, 2069–2073 (2003)

    CAS  Article  Google Scholar 

  30. 30

    Hardie, R. C. Regulation of TRP channels via lipid second messengers. Annu. Rev. Physiol. 65, 735–759 (2003)

    CAS  Article  Google Scholar 

  31. 31

    Melyan, Z., Tarttelin, E. E., Bellingham, J., Lucas, R. J. & Hankins, M. W. Addition of human melanopsin renders mammalian cells photoresponsive. Nature doi:10.1038/nature03344 (this issue)

  32. 32

    Panda, S. et al. Illusion of the melanopsin signaling pathway. Science 307, 600–604 (2005)

    CAS  Article  ADS  Google Scholar 

  33. 33

    Isoldi, M. C., Rollag, M. D., de Lauro Castrucci, A. M. & Provencio, I. Rhabdomeric phototransduction initiated by the vertebrate photopigment melanopsin. Proc. Natl Acad. Sci. USA 102 (4), 1217–1221 (2005)

    CAS  Article  ADS  Google Scholar 

Download references

Acknowledgements

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.

Author information

Affiliations

Authors

Corresponding author

Correspondence to David M. Berson.

Ethics declarations

Competing interests

The authors declare that they have no competing financial interests.

Supplementary information

Supplementary Data

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)

Supplementary Methods

Additional details on cell culture, expression of melanopsin, electrophysiological and pharmacological techniques, photic stimulation methods, spectral analysis, and calcium imaging. (DOC 38 kb)

Supplementary Figure 1

Light evokes calcium responses in HEK293-TRPC3 cells that express melanopsin but not EGFP. (DOC 275 kb)

Supplementary Figure 2

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)

Supplementary Figure 3

Cumulative irradiance-response functions at the six wavelengths tested, pooled across all recorded cells. (DOC 124 kb)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

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

Download citation

Further reading

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.