(i) Ciliary opsins

In addition to being expressed in ciliary photoreceptor cells characterized by an extended cilium, ciliary opsins also share some similarities in their phototransduction mechanism. Although they can produce different responses, all opsins in this group seem to function through signalling cascades that alter the concentration of cyclic nucleotides.

Vertebrate opsin/encephalopsin. The vertebrate opsin/encephalopsin subfamily consists of vertebrate opsins and encephalopsins.

Vertebrate opsins. The vertebrate opsin group comprises visual and non-visual opsins in vertebrates. Phylogenic analysis of vertebrate visual opsins reveals that they can be further subdivided into five subgroups consisting of four cone opsins and one rod opsin group, which are distinguished by their spectral sensitivity (Okano et al. 1992b). The S group consists of cone opsins that absorb UV or violet light, the M1 group absorbs blue light, the M2 group absorbs green light and the L group absorbs red or green light. The rod opsin group, denoted Rh, absorbs green/blue light and is a sister group to the M2 group. Cone and rod opsins function through the well-characterized Gt signalling pathway (figure 1). In addition to their role as ocular photoreceptor molecules, vertebrate visual opsins are also expressed in non-visual photoreceptor cells, such as the pineal photoreceptor cells; however, their role and/or contribution in non-visual photoreception remains unknown (Wada et al. 1998; Mano et al. 1999).

The non-visual opsins in the vertebrate opsin subfamily consist of pinopsins, parapinopsins, VA (vertebrate ancient) opsins and parietopsins. They are opsins that closely resemble vertebrate visual opsins, with more than 40 per cent identity. Non-visual opsins are presumed to be involved in light-dependent physiological phenomena, such as photic regulation of circadian rhythms, photoperiodicity and body colour change. Pinopsins are found in the pineal organ of avian species, reptiles and amphibians, where they may play a role in its regulation, but they seem to be absent from teleosts and mammals (Okano et al. 1994; Max et al. 1995; Taniguchi et al. 2001). Parapinopsins have been found in the photosensitive pineal and parapineal organs of jawless fish, teleost fish and amphibians (Blackshaw & Snyder 1997; Koyanagi et al. 2004). VA opsins were initially identified in salmon and they were named VA opsins because they seem to have diverged early in vertebrate opsin evolution (Soni & Foster 1997). They are localized in the inner retina and the brain in teleosts. Isoforms of VA opsins called VAL opsins have been reported, characterized by the extension of their carbonyl terminus (Kojima et al. 2000; Moutsaki et al. 2000; Minamoto & Shimizu 2002). A recent study reported that VAL opsins are duplicated in the teleost lineage and that the two copies of VAL opsin have a differential expression, suggesting that they have different physiological roles (Kojima et al. 2008).

Lizards and other non-mammalian vertebrates have been known to possess a photoreceptive organ on the top of their head, called the parietal eye, complete with a cornea, a lens and a retina. The opsin expressed in the ‘third eye’, named parietopsin, is closely related to the vertebrate visual opsins, with approximately 40 per cent identity to parapinopsins and VA opsins (Su et al. 2006). Interestingly, the photoreceptor cells of the parietal eye possess two signalling pathways, which are activated in response to different light. Blue light causes hyperpolarization through the pinopsin–gustducin signalling pathway, and green light causes depolarization through the parietopsin–Go signalling pathway.

Encephalopsins: There are two main groups in this opsin group: encephalopsins (or panopsins)/tmt opsins and Platyneresis c-opsin/pteropsins. Encephalopsins were initially identified in mammals, and their name was derived from their strong expression in the brain and testes (Blackshaw & Snyder 1999). However, subsequent studies showed that this opsin is also widely expressed, although weakly, in non-photoreceptive tissue, such as the heart, lung, liver, kidney, skeletal muscle and pancreas, as well as in the retina (Halford et al. 2001). Because of its wide distribution, it has been suggested that encephalopsin would be better named panopsin. Teleost multiple tissue (tmt) opsin is a homologue of mammalian encephalopsin, found in teleost fish, widely distributed in neuronal and non-neuronal tissue, where it has been proposed as the photopigment that regulates photic entrainment of peripheral clocks (Moutsaki et al. 2003).

A homologue of encephalopsin and tmt opsin was identified in ciliary photoreceptor cells in the brain of the marine rag worm, Platyneresis (Arendt et al. 2004). This finding surprised researchers because it was the first ciliary opsin to be identified in protostomes. Moreover, an orthologue (orthologues are genes in different species that originated from a common ancestral gene through speciation) called pteropsin was reported in insects (Hill et al. 2002; Velarde et al. 2005). Pteropsins and Platyneresis c-opsin have great similarity to vertebrate opsins, and they also have three common introns with vertebrate opsins, indicating a close relationship with vertebrate opsins. They are expressed in insect brains and not in their eyes. Interestingly, this lineage of opsins was lost in Drosophila, a phenomenon that delayed its discovery.

Go opsins. Go opsins have been identified in molluscs and in the chordate amphioxus, but they are not present in humans, mice, zebrafish or fruitfly (Kojima et al. 1997; Koyanagi et al. 2002). Like vertebrate opsins/encephalopsins, they are expressed in ciliary photoreceptor cells. Light stimulation of these ciliary photoreceptor cells results in an increase in cGMP, probably through the activation of membrane GC (guanylyl cyclase), that subsequently opens K⁺ selective channels and thus causes a hyperpolarization response (Gomez & Nasi 2000).

Gs opsins. The most recently described opsin subfamily is that of opsins found in cnidarians, including the sea anemone, hydra and jellyfish (Plachetzki et al. 2007; Suga et al. 2008). More recently, the box jellyfish opsins, which also cluster in this subfamily, have been shown to signal through the Gs signalling pathway, involving an increase in cAMP (Koyanagi et al. 2008b). These opsins are also expressed in ciliary photoreceptor cells.

(ii) Rhabdomeric opsins

Rhabdomeric opsins are localized in the microvilli of rhabdomeric photoreceptor cells, which are morphologically different from ciliary photoreceptor cells. These opsins transmit light signals through the G protein Gq subgroup, involving phospholipase C (PLC) (Terakita et al. 1993; Lee et al. 1994). Dissociated Gqα binds to its effector enzyme, PLCβ, and activates it. The substrate of PLC is a membrane phospholipid, PIP₂, which is separated into two potential messengers: DAG (diacylglycerol) and IP₃ (inositol 1,4,5-tris-phosphate). Exactly how these messengers act is still unknown, but similar to the vertebrate visual signalling cascade, they act on a membrane channel. However, contrary to the vertebrate visual CNG (cyclic nucleotide gated) channel, the invertebrate visual TRP (transient receptor potential) channel opens in response to light stimuli, creating a depolarization response. In contrast to the vertebrate visual opsins, most of the invertebrate Gq opsins produce a thermally stable active state and the photo-activated molecule can be reconverted to the ground state by absorption of a second photon. The biochemical study of these opsins has been delayed because they are difficult to express in cultured cells, and only recently did the exogenous expression of these opsins became possible (Terakita et al. 2008). Also recently, the crystal structure of squid rhodopsin was solved (Murakami & Kouyama 2008). These recent breakthroughs should catalyse our understanding of these opsins.

Invertebrate Gq opsins. The invertebrate Gq opsin family contains the arthropod and mollusc visual opsins, as well as melanopsins. Like the vertebrate visual opsins, arthropods possess a well-documented colour vision, supported by Gq opsins tuned to a specific colour (Briscoe & Chittka 2001; Koyanagi et al. 2008a). Melanopsins are found in vertebrates, but they closely resemble invertebrate visual opsins (Provencio et al. 1998, 2000). Initially identified in amphibians, they have been subsequently reported in all vertebrate classes. Melanopsins are the primary photoreceptor molecules for non-image forming function such as the photo-entrainment of the circadian clock and pupillary constriction in mammals (Hattar et al. 2003; Lucas et al. 2003; Panda et al. 2003). It has recently been shown that melanopsin also participates in visual tasks, by regulating optic inputs from photoreceptor cells, according to the circadian phase, i.e. the time of the day (Dacey et al. 2005; Barnard et al. 2006).

(iii) Photoisomerases

Photoisomerases are stereospecific isomerases that bind all-trans-retinal and generate 11-cis-retinal. As their biological role is not to initiate a signalling cascade, they are not coupled to a G protein and thus they do not generate a signalling cascade. The photoisomerase opsin family consists of retinochrome, RGR opsins and peropsins.

Retinochrome/RGR opsins. Retinochrome is the retinal isomerase that supplies 11-cis-retinal to the visual cycle in molluscs (Hara & Hara 1967). RGR opsin expression is confined to the retinal pigment epithelium (RPE) and Müller cells of vertebrates, and to the brain of a chordate ascidian (Jiang et al. 1993; Nakashima et al. 2003). Its endogenous chromophore is the all-trans form of retinal and it can photoisomerize it to 11-cis in a stereospecific manner, although the resulting 11-cis-retinal decays thermally back to all-trans-retinal (Hao & Fong 1999). It would appear that rather than supplying 11-cis-retinal to other opsins, RGR opsins regulate retinoid traffic in RPE in a light-dependent manner (Radu et al. 2008).

Peropsins. Peropsins have been reported in vertebrates and the chordate amphioxus (Sun et al. 1997; Koyanagi et al. 2002). Phylogenetically, they are closely related to photoisomerases, and their localization in the RPE suggests that they may also function as photoisomerases. In fact, they can bind all-trans-retinal and photoisomerize it to 11-cis-retinal (Koyanagi et al. 2002). However, contrary to other photoisomerases, peropsin possesses sequence motifs conserved among family-A GPCRs. The highly conserved D/ERY triad at the cytoplasmic surface and the NPXXY motif at H7 are important for the activation of the G protein. The presence of these structural features suggests that peropsin may bind and activate a G protein, although its physiological significance is unclear.

(i) Evolution of the counterion

As mentioned earlier, the retinal attaches to the opsin in H7 in the transmembrane domain through a protonated Schiff base linkage. Although protonation of the Schiff base is necessary for visible light reception, a positive charge in the hydrophobic transmembrane environment is highly unstable. Opsins counter this problem by providing a negatively charged amino acid, a counterion that stabilizes the positive charge of the protonated Schiff base. There are two tentative sites that can serve as a counterion in opsins: 113 at H3 in the transmembrane domain, and 181 at the ECL2 (extracellular loop 2) between H4 and H5 (figures 2 and ​and5).5). The residue at 181 is a conserved glutamic (aspartic) acid among all opsin subfamilies, whereas 113 vary between them.

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Figure 5.

The counterion of opsins. (a) Vertebrate visual and non-visual opsins have a negatively charged glutamic acid at 113 in H3 that functions as the counterion for the positive charge of the protonated retinal Schiff base. (b) The counterion of Go/Gs/Gq opsins and photoisomerases is E181.

Vertebrate opsins and encephalopsins generally have a glutamic acid on both 113 and 181. Mutations at E113 in vertebrate visual opsins result in a big blue shift in the absorbance maxima of the pigment, making it UV light sensitive, an indication that the Schiff base is deprotonated (Sakmar et al. 1989). On the other hand, mutations at E181 do not disturb the absorbance maxima. These experiments indicate that E113, and not E181, is the one that serves as the counterion in the vertebrate opsins group.

The counterion of other opsins was first identified in the mollusc photoisomerase retinochrome (Terakita et al. 2000). In this opsin, 113 is occupied by residues that do not possess a negative charge, methionine or histidine, while 181 is occupied by the conserved glutamic acid. Site-directed mutagenesis showed that mutations at 113 do not disturb the absorbance spectra, whereas mutations induced at E181 result in a deprotonated Schiff base, indicating that E181 serves as the counterion in photoisomerases.

In contrast to easily expressed vertebrate opsins and retinochrome, invertebrate Gq opsins are not readily expressed in cultured cells, and their functional characterization has been delayed.

Although there is no direct experimental evidence for the Gq opsin counterion, Gq, Go, peropsins and neuropsins all have a tyrosine or a phenylalanine residue at 113. Furthermore, mutational experiments on Go opsins and peropsins have shown that they behave similar to retinochrome (Terakita et al. 2004). Mutations at 113 do not cause deprotonation of the Schiff base, whereas mutations at E181 cause spectral changes characteristic of Schiff base deprotonation.

Taken together, these experiments show that only vertebrate opsins have a counterion at 113, whereas all other opsins seem to have a counterion at the conserved glutamic acid at 181 (figure 5). Interestingly, the counterion of Go opsins can be artificially moved from 181 to 113. As mentioned earlier, mutations at 181 in Go opsins cause spectral changes owing to the deprotonation of the Schiff base. However, a second mutation introducing a glutamic acid in 113 reverses the deprotonation, showing that the glutamic acid at 113 can act as a counterion (Terakita et al. 2004). This experiment proved that 113 can serve as a counterion in addition to its original counterion at 181, which is in contrast with the vertebrate opsins, where only 113 can serve as the counterion. Moreover, these experiments also revealed that the glutamic acid at 113 in Go opsins could serve as a counterion in the resting state but not in the signalling state. It seems that an ancestral pigment had a counterion at 181, and vertebrate opsins recruited E113 as a novel counterion. Acquisition of the novel counterion at 113 relaxed the function of 181 as a counterion, probably causing rearrangements in ECL2 that further prevented it from functioning as a counterion.

Detailed analyses of E113 in vertebrate visual opsins have revealed some interesting functions of this residue, in addition to its function as the counterion for visible light sensing. E113 has been shown to serve as a ‘molecular switch’ which suppresses rhodopsin activation in the absence of light (Robinson et al. 1992). The attraction of the positive charge of the protonated K296 and the negative charge of the deprotonated E113 maintain the receptor in an inactive resting state (figure 5). Light absorption diminishes this interaction and the proton of the Schiff base is transferred to E113, triggering the formation of a hydrogen network that activates the receptor (Jäger et al. 1994). Therefore, vertebrate visual pigments have a photoproduct with a deprotonated Schiff base, which contributes to the efficient activation of the G protein.

However, the newly acquired counterion in vertebrate opsins did not immediately act as a molecular switch. The analysis of the photoproducts of vertebrate opsins shows that only vertebrate visual opsins have a deprotonated photoproduct, suggesting that parapinopsins and encephalopsins behave more like invertebrate opsins, despite their sequence similarity to vertebrate visual opsins. It is believed that during the course of vertebrate visual opsin evolution, E113 acquired the capacity to act as a molecular switch. Moreover, it seems that E113 may have an additional role in enhancing light sensitivity, by increasing the isomerization efficiency upon light absorption (Tsutsui et al. 2008).

After E113 took over the role of counterion, the selective pressure on E181 must have decreased, allowing different amino acids at this position. An interesting mutation is observed in cone opsins, which are sensitive to red light, i.e. they absorb light at longer wavelengths. The position 181 is occupied by histidine instead of the highly conserved glutamic acid (Nathans et al. 1986; Kuwata et al. 1990). These unusual substitutions are considered to be special adaptations for the absorption of red light. It turns out that H181 is part of a chloride binding site, which causes the chromophore to maximally absorb light of longer wavelength (Wang et al. 1993). This adaptation was possible because of the counterion displacement from 181 to 113, which freed 181 from its structural constraints as the counterion, allowing it to acquire a new function.