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Parietal-Eye Phototransduction Components and Their Potential Evolutionary Implications

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Science  17 Mar 2006:
Vol. 311, Issue 5767, pp. 1617-1621
DOI: 10.1126/science.1123802

Abstract

The parietal-eye photoreceptor is unique because it has two antagonistic light signaling pathways in the same cell—a hyperpolarizing pathway maximally sensitive to blue light and a depolarizing pathway maximally sensitive to green light. Here, we report the molecular components of these two pathways. We found two opsins in the same cell: the blue-sensitive pinopsin and a previously unidentified green-sensitive opsin, which we name parietopsin. Signaling components included gustducin-α and Gαo, but not rod or cone transducin-α. Single-cell recordings demonstrated that Go mediates the depolarizing response. Gustducin-α resembles transducin-α functionally and likely mediates the hyperpolarizing response. The parietopsin-Go signaling pair provides clues about how rod and cone phototransduction might have evolved.

Lizards and some other lower vertebrates have a third eye (parietal eye) (1) in addition to the two lateral eyes. This eye may mediate the global detection of dawn and dusk (1, 2) instead of conventional image-forming vision. The parietal-eye photoreceptors resemble rods and cones in morphology, but they show chromatic antagonism (a unique feature among all known photoreceptors) consisting of a hyperpolarizing light response most sensitive to blue light and a depolarizing light response most sensitive to green light (2). The hyperpolarizing response is produced, as in rods and cones, by the activation of a guanosine 3′,5′–cyclic monophosphate (cGMP)–phosphodiesterase that lowers the cGMP concentration and closes cyclic nucleotide-gated (CNG) channels (3, 4). The depolarizing response, on the other hand, is produced by the inhibition of the same phosphodiesterase, elevating cGMP and opening CNG channels (3, 4). We cloned the molecular components underlying this antagonism.

By screening a parietal-eye cDNA library from the side-blotched lizard (Uta stansburiana) for vertebrate opsins (5), we found pinopsin, a blue-sensitive pigment first identified in chicken pinealocytes (6, 7). In addition, we found a previously unidentified opsin, which we named parietopsin (fig. S1). We also found orthologs of parietopsin in fish and frog DNA databases. The conserved glutamate constituting the counterion for the protonated Schiff-base in typical opsins is replaced in parietopsin by glutamine (Gln103), indicating that parietopsin uses another conserved glutamate (probably Glu171) as the counterion (8). This feature appears to be a characteristic of evolutionarily ancient vertebrate opsins (8). In pairwise alignments, parietopsin showed the highest degree of identity (∼40%) to parapinopsin, an ancient opsin identified in the fish parapineal organ (9) (table S1). However, a phylogenetic analysis including lizard parietopsin and its orthologs demonstrated that parietopsins defined a distinct opsin subfamily (Fig. 1A). When expressed in human embryonic kidney (HEK) 293 cells, parietopsin had a λmax at 522 nm (Fig. 1B), indicating that it is a green-sensitive pigment.

Fig. 1.

Parietal-eye opsins. (A) Phylogenetic analysis of parietopsin and other vertebrate opsins. The tree was constructed by the Neighbor-Joining algorithm with bacteriorhodopsin as an outgroup (5). Bootstrap probability (%), which is a statistical evaluation of the robustness of the grouping, is shown when ≥50. Parietopsins defined a distinct opsin subfamily with a very strong bootstrap support (100%). Opsins identified in this study are shown in red. RH, rhodopsin; SWS, short wavelength–sensitive opsin; LWS, long wavelength–sensitive opsin; MWS, medium wavelength–sensitive opsin; VA, vertebrate ancient opsin; RGR, retinal pigment epithelium G protein–coupled receptor; OPN5, neuropsin. (B) Absorption spectrum of purified lizard parietopsin expressed in HEK293 cells. It showed a λmax at 522 nm at pH 6.5 with 11-cis-retinal as chromophore, in darkness (5) (SOM text). (Inset) The full dark spectrum of parietopsin. The ratio of the protein absorbance at 280 nm to the λmax of parietopsin is 7.4. (C) Sequence alignments of the second and third cytoplasmic loops of the lizard opsins identified in this study. Identical and similar residues are shaded in dark and light gray, respectively. PtOP, parietopsin; POP, pinopsin. (D) Confocal images of parietal-eye sections double-stained with antibodies against parietopsin (green) and pinopsin (red). Nuclei were counterstained with 4′,6′-diamidino-2-phenylindole (DAPI) in blue. The staining of parietopsin antiserum beneath the ganglion cell layer (GCL) was nonspecific because it was also seen with the preimmune serum. The merged image (right) shows that parietopsin and pinopsin colocalized in the outer segments of the photoreceptors. Scale bar, 5 μm. OS, outer segment; PRL, photoreceptor layer.

For comparison, we also identified five lateral-eye (i.e., rod and cone) opsins in the same lizard (5). Polymerase chain reaction (PCR) analyses indicated that the lateral and parietal eyes expressed nonoverlapping sets of opsins [fig. S2 and supporting online material (SOM) text]. In rod and cone opsins, the second and third cytoplasmic loops are highly conserved, reflecting their importance in specific interactions with the downstream G protein, transducin (10). Pinopsin, but not parietopsin, shared these homologous sequences (Fig. 1C). Double immunolabeling with specific antibodies showed that pinopsin and parietopsin colocalized in the same photoreceptor outer segment, where phototransduction takes place (Fig. 1D; see also fig. S3 for antibody specificities). Thus, pinopsin and parietopsin appear to drive the hyperpolarizing and depolarizing responses, respectively.

In the chicken, pinopsin colocalizes with rod transducin-α in pinealocytes (11) and activates it in the light (11, 12), suggesting that the two are functionally coupled in these cells. However, we did not find rod or cone transducin-α in the parietal-eye cDNA library (although we did find them in the lateral-eye cDNA library). Instead, we found gustducin-α, the G protein mediating bitter and sweet transductions in taste buds (13, 14). Sequence alignments of the lizard gustducin-α and rod and cone transducin-α proteins showed 80% identities among all three (fig. S4); the degrees of sequence identities are similar in the rat (13). With reverse transcription (RT)–PCR, we confirmed the same gustducin sequence in lizard tongue. RT-PCR also showed that the gustducin-α message was absent in the lateral eyes, and the rod and cone transducin-α messages were absent in the parietal eye (15), confirmed by immunocytochemistry with antibodies against the cloned gustducin-α and cone transducin-α, respectively (Fig. 2, A and B).

Fig. 2.

G proteins and other phototransduction components. (A to B) Immunocytochemistry of parietal-eye retina and lateral-eye retina with the use of specific antibodies against cone transducin-α (Gt2α) and gustducin-α (Ggustα). Parietal-eye photoreceptors expressed Ggustα but not Gt2α. Conversely, Gt2α, but not Ggustα, was expressed in the lateral eyes. Most lateral-eye photoreceptors were labeled by the Gt2α antibody because the retina of the side-blotched lizard is cone dominant. A monoclonal antibody, TF15, that recognizes both Ggustα and Gt1α/Gt2α was used as a positive control. Merged images show that TF15 labeled Ggustα in the parietal eye and Gt1α/Gt2α in the lateral eye. (C to E) Double-immunostaining of parietal-eye sections. Ggustα was labeled by TF15, as shown in (A). (C) Gαo labeling in the outer segments overlapped with Ggustα. Likewise, Gβ3 and Gγc (D), as well as PDEγ (E), also colocalized with Ggustα in the parietal-eye photoreceptors. Gβ3 and Gγc, similar to Ggustα, were expressed throughout the photoreceptor, whereas PDEγ labeling was found mainly in the outer segment. The specificities of the Gβ3 and Gγc antibodies we used were verified in the lateral-eye retina, where their labelings colocalized with that of cone transducin-α in most photoreceptors (15). OS/IS, outer segment/inner segment; ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer; PRL, photoreceptor layer. Scale bars, 5 μm [(A) and (C) to (E)] and 10 μm (B).

We also found Gαo in the parietal-eye cDNA library. A polyclonal antibody (K-20, Santa Cruz Biotechnology) that recognized Gαo in lizard lateral-eye ON-bipolar cells (16) (positive control; fig. S5) but did not cross-react with transducin-α in rods or cones also labeled the parietal-eye photoreceptors. Double-immunolabeling with K-20 and a monoclonal antibody that recognizes lizard gustducin-α (TF15, CytoSignal; Fig. 2A) confirmed the colocalization of gustducin-α and Gαo in the same photoreceptor outer segment (Fig. 2C).

The similarities in structure and function between gustducin-α and transducin-α (13, 17, 18), together with the similarity between pinopsin and rod and cone opsins, suggest that pinopsin couples to gustducin to produce the hyperpolarizing light response. If so, parietopsin likely couples to Go to produce the depolarizing light response. For functional verification, we did whole-cell recordings from isolated parietal-eye photoreceptors. At a holding potential of –45 mV [near the membrane potential in darkness (2)], cGMP in the whole-cell pipette induced an inward current in darkness by opening CNG channels (3, 4). The 100 μM cGMP induced a much smaller current (36 ± 5 pA, mean ± SEM, n = 27) than the 150 μM cGMP (118 ± 28 pA, n = 6) (Fig. 3A, top and bottom), indicating that the current induced by 100 μM cGMP was far from saturated, attributable to high endogenous phosphodiesterase activity (4). With 100 μM cGMP, a white flash elicited a biphasic response consisting of an outward current followed by an inward current (Fig. 3B, top left). A green (520-nm) flash, however, elicited only an inward current, and a blue (480-nm) flash elicited only an outward current (Fig. 3B, middle and bottom left), corresponding to a membrane depolarization and hyperpolarization, respectively, under current clamp (2). When the pipette also contained 20 μM mastoparan, a peptide that activates Go but has little effect on transducin (19), the inward current induced by 100 μM cGMP increased to 92 ± 16 pA (n = 18) (Fig. 3A, middle and bottom). Also, a white flash in this case elicited only an outward current (Fig. 3B, top right). Correspondingly, a green flash elicited no response, whereas a blue flash still elicited an outward current (Fig. 3B, middle and bottom right). Finally, the inactive mastoparan analog Mas-17 (20) affected neither the cGMP-induced current in darkness (Fig. 3A, bottom) nor the light responses (15). These results suggest that Go inhibits the phosphodiesterase to produce the depolarizing light response (4), so that its activation by mastoparan in our experiments increased the cGMP-induced inward current in darkness and occluded the depolarizing light response. Collectively, a white flash elicited a response with an inward-current component in 24 out of 25 cells in the absence of mastoparan, but it did so in only 1 out of 13 cells in the presence of mastoparan. Correspondingly, a white flash–induced response showed an outward-current component in 14 out of 25 cells without mastoparan [the hyperpolarizing pathway is less sensitive to light (2)] and still showed the outward-current component in all of 13 cells with mastoparan. Thus, the effect of mastoparan was quite specific. The inhibition of the phosphodiesterase by Go could be direct or indirect (SOM text).

Fig. 3.

Whole-cell recordings from isolated parietal-eye photoreceptors. (A) Inward current induced in darkness by 100-μM cGMP in the whole-cell pipette (membrane holding potential = –45 mV) in the absence (top) or presence (middle) of mastoparan, a Go-activating peptide. The mastoparan concentration (20 μM) was empirically chosen in order to selectively activate Go and at the same time to minimize nonspecific effects (19, 26). The arrow indicates the time point at which whole-cell recording started. Collective results are shown at the bottom. Mas-17, an inactive form of mastoparan, was used as a negative control. (B) Light responses recorded after the cGMP-induced current reached steady state. (Left) Top, middle, and bottom panels are from the same cell. In the absence of mastoparan, the response to a white flash was biphasic (top), composed of a green-sensitive inward current and a blue-sensitive outward current, as revealed by the subsequent light responses at 520 nm (middle) and 480 nm (bottom). In the top panel, the cGMP-induced current before the flash was –44 pA. (Right) Similar experiment from another cell, but with mastoparan. In the presence of mastoparan, the response to a white flash showed only an outward-current component (top). A 520-nm flash produced no response (middle), whereas a 480-nm flash still produced an outward-current response (bottom) in this cell. In the top panel, the cGMP-induced current before the flash was –92 pA.

By immunocytochemistry, we also found in the parietal-eye photoreceptor Gβ3 and Gγc (i.e., cone transducin-βγ) (21, 22), but not Gβ1 and Gγ1 (rod transducin-βγ), despite their positive labelings in the lateral eyes. The labeling of Gβ3 and Gγc colocalized with that of gustducin-α (Fig. 2D). Further cDNA library screenings identified the catalytic subunit (PDEα′) and the inhibitory subunit (PDEγ) of the cone cGMP phosphodiesterase (23), which also colocalized with gustducin-α in the outer segment of the parietal-eye photoreceptor (Fig. 2E). Finally, in the parietal eye, we found clones for the rod CNG channel α subunit (CNGA1) and the guanosine triphosphatase (GTPase)–activating protein (GAP) complex (RGS9 and Gβ5L) common to rods and cones, proteins critical for response termination by promoting the GTPase activity of transducin (24). Previous electrical recordings also corroborate that the CNG channel on the parietal-eye photoreceptor is more similar to that of rods than that of cones (3).

The emerging picture is as follows (fig. S6): The hyperpolarizing light response is mediated by the blue-sensitive pinopsin, which activates a cGMP-phosphodiesterase by means of gustducin to lower cGMP concentration and close CNG channels. The depolarizing light response is mediated by the green-sensitive parietopsin, which inhibits the phosphodiesterase by means of Go to elevate cGMP and open CNG channels. In conjunction with this chromatic antagonism, the parietal-eye photoreceptor synapses directly on ganglion cells (1). Thus, unlike in the lateral eyes where elaborate neuronal circuitry involving bipolar, horizontal, and amacrine cells performs substantial visual-signal processing (including chromatic antagonism), information processing in the primitive parietal eye takes place at least partly in the photoreceptors themselves, with the use of two pigments and two G proteins. Additionally, the molecular identities of the phototransduction components reported here may shed light on the evolutionary status of this photoreceptor. A clue comes from Go, which also mediates phototransduction in the scallop hyperpolarizing photoreceptor (an invertebrate ciliary photoreceptor) (25) by coupling the visual pigment SCOP2 to the activation of a guanylate cyclase and leading to the opening of a cGMP-gated potassium channel (25, 26). Similar to parietopsin, SCOP2 appears to be an ancient opsin that has diverged early in opsin evolution (25). Thus, a Go-mediated phototransduction mechanism appears ancient. Moreover, although gustducin, transducin, and Go all belong to the same G protein subfamily (Gi), Go appears to be the most ancient because it is common to vertebrates and invertebrates. Accordingly, we propose the following evolutionary lineage of phototransduction mechanisms in ciliary photoreceptors, assuming that vertebrate and invertebrate (including the scallop ciliary photoreceptor) share one common precursor (27, 28). A Go-mediated phototransduction pathway might already be present in the ciliary photoreceptors of early coelomates, the last common ancestor of lizard (vertebrate) and scallop (mollusk), because both have this pathway. Later, the ancestral vertebrate photoreceptor acquired a second G protein, either gustducin or transducin, for chromatic antagonism and perhaps other purposes (SOM text). The parietal photoreceptor evolved subsequently and retained these ancestral features. The cones, rods, and light-sensitive pinealocytes [which are mechanistically identical to rods and cones in phototransduction (29, 30)], on the other hand, inherited only the gustducin/transducin-mediated pathway.

Supporting Online Material

www.sciencemag.org/cgi/content/full/311/5767/1617/DC1

Materials and Methods

SOM Text

Figs. S1 to S6

Tables S1 and S2

References

References and Notes

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