Opsin mediates circadian clock

Research shows that melanopsin acts as a bistable pigment in vertebrate ganglion

By | January 28, 2005

Scientists report proof this week that melanopsin protein mediates the circadian clock. The findings, published in Nature and Science, confirm the theory that melanopsin triggers photosensitivity and describe surprising similarities between vertebrate and invertebrate cell phototransduction cascades.

"It's almost a paradigm, thinking about vertebrate and invertebrate opsins in this way," Satchin Panda, author of the Sciencestudy, told The Scientist. "We see that light sensing and time- keeping is conserved [through evolution]." In his study, published online in advance of print, Panda, of the Salk Institute in La Jolla, Calif., describes melanopsin's ability to active the G-protein signaling pathway in Xenopus oocytes.

The papers follow the publication earlier this month in PNAS of a study that found that Xenopus' dermal melanosomes use a phosphoinositide signaling pathway similar to that of invertebrates. That study was functional confirmation of earlier molecular analyses, which showed that Xenopus melanophores and ipRGCs may have evolved from invertebrate photoreceptor cells—known as rhabdomeres—as opposed to ciliary photoreceptors commonly used by vertebrates.

In Nature, a team led by David Berson, professor of neuroscience at Brown University, introduced mouse melanopsin into a human kidney cell line (HEK293) that expressed stable TRPC3 channels. When they transfected the cells with green fluorescent reporter protein tagged with melanopsin, they found that light exposure opened the TRPC3 channels, depolarized the cell membrane, and released intracellular calcium—a response similar to those found in studies of isolated rat intrinsically photosensitive retinal ganglion cells (ipRGCs) and studies of rodless, coneless rodents. Measurements of the HEK293 cells' spectral sensitivity found a threshold of 479 nm, similar to that of ipRGCs.

HEK293 cells demonstrated continuous charge, even though they lacked a retinaldehyde chromophore that binds melanopsin, the protein thought necessary to initiate G-protein cascade and ultimately the light response. That finding suggests, Berson told The Scientist, that kidney cells may possess intrinsic light-triggered retinoid binding proteins and isomerases.

In another paper in Nature, Rob J. Lucas, of the University of Manchester, and M.W. Hankins, of Imperial College, London, and colleagues performed similar experiments using human melanopsin and mouse neural cells. They transfected Neuro-2a blastoma cells with melanopsin and green fluorescent reporter protein. When they added 9-cis retinaldehyde, the cells showed spectral sensitivity in the range of 360–430 nm.

This range, Lucas said, falls below the expected action spectrum for ipRGCs, and reasons for the result remain unclear. "It may be something about [these] cells which change the spectral sensitivity of melanopsin proteins," Lucas told The Scientist, such as "the interaction between the protein and the retinaldehyde chromophore or [due to] the cellular environment." Ignacio Provencio, of the University of Virginia, a co-author of the Berson Nature study, said that human and mouse melanopsin may also be different.

Addition of all-trans retinaldehydes only produced significant current when exposed to longer light wavelengths, results that suggest, write the authors, that melanopsin functions ideally as a photoreceptor in the presence of short light wavelength and cis-retinoids.

The cells did not produce current at 540 nm—a wavelength outside the response range of cis-isomers—and no other known photoisomerases were found in the Neuro-2a cells. That suggests that the all-trans retinoids photoisomerize, or regenerate into cis-conformation. And, according to several authors of the Nature and Science papers, melanopsin's amino acid sequence, a key element for chromophore binding, more closely resembles invertebrate opsins.

For these reasons, researchers think that vertebrate melanopsin functions as a bistable pigment—absorbing shorter wavelength light with the aid of cis-retinaldehyde, and regenerating bleached chromophores via photoisomerization using longer light wavelengths. That allows continuous light absorption by direct conversion of retinoid, rather than having photons go through a pathway of the vertebrate's retinal pigment epithelium. Only invertebrates were thought to be able to regenerate chromophores in this way. Berson jokes: "[It] looks like there's a little bit of fly eye wrinkled in [the] mammalian retina."

"Their results have good explanatory power for how [melanopsin] has been evolutionary derived and what its job might be," said Lawrence Morin of the State University of New York at Stony Brook, who did not participate in any of the studies.

Berson's group will now focus on how the G-protein signaling cascade works in vertebrates. Lucas and colleagues will explore melanopsin's short wavelength shift and how cell environment may play a role in spectral sensitivity using neural primary culture.

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