Ординатура / Офтальмология / Английские материалы / The Retina and its Disorders_Besharse, Bok_2011

with bovine rhodopsin is important for intracellular trafficking of this rhodopsin, and its deletion does not affect G protein activation. Like other GPCRs, squid rhodopsin forms a disulfide bridge between Cys108 and Cys186 for proper folding of the E-II loop. The NPXXY and (D/E)R (Y/W) regions of squid rhodopsin are similar to those of bovine rhodopsin. However, Val in the NPXXY motif, and Glu in the (D/E)R(Y/W) motif of bovine rhodopsin are replaced by Met and Asp, respectively, in squid rhodopsin (Figure 6(b)). Their structural similarity implies that the functional difference between vertebrate and invertebrate rhodopsins might be due to specific interactions between retinal and the amino acid sequence of the retinal-binding site. These binding sites are compared in Figures 6(c) and 6(d). In squid rhodopsin, the highly conserved Glu180 is too

far away from the retinal-binding pocket and Asn185 is located between Glu180 and the Schiff base. Asn185 is

assumed to move after photoisomerization of retinal, to mediate an indirect interaction between Glu180 and the retinal Schiff base. In the dark state, either Asn87 or Tyr111, which is highly conserved among all invertebrates, might act as a hydrogen-binding partner (counter ion) for the Schiff base. These residues are replaced by Gly89 and Glu113 in bovine rhodopsin.

Signaling Cycle

After photoactivation, an active meta II state of rhodopsin triggers the activation of transducin (Gt protein). This form of rhodopsin is capable of activating Gt proteins during the relatively prolonged period of its activation. Accordingly, rhodopsin activity is regulated by its phosphorylation, a common feature among many GPCRs. This regulatory process is also important for rod cells to recover their responsiveness during dark adaptation. Desensitization of rhodopsin involves two steps: (1) phosphorylation of meta II, reducing the rate of transducin activation and (2) binding of arrestin to meta II, completely ending transducin activation by rhodopsin (Figure 7). This phosphorylation is carried out by rhodopsin kinase, a specific kinase also known as GRK1. Rhodopsin kinase phosphorylates specific serines in the rhodopsin C-terminal sequence. However, upon light illumination, GRK1 is

released from recoverin and phosphorylates rhodopsin at multiple sites. Ser343, Ser338, and Ser334, located in the

C terminal domain on the cytoplasmic side of rhodopsin, are the main sites for this phosphorylation. Some threonine residues in this domain are also phosphorylated. Phosphorylation of this cytoplasmic domain reduces the ability of the Gt protein to bind to meta II, but it does not completely stop Gt activation. By binding to phosphorylated-meta II, arrestin prevents the interaction of Gt with meta II, and thus completely terminates Gt activation. At least three phosphorylated sites are required for high-affinity binding

Arrestin

P PP

Arrestin

All-trans-retinal

Arrestin

Pi-meta II/arrestin

Figure 7 Interaction of rhodopsin with partner proteins. Phototransduction starts with the absorption of light by rhodopsin that causes photoisomerization of 11-cis-retinal to all-trans-retinal. Photoisomerization of this chromophore induces conformational changes in rhodopsin leading to formation of meta II, the signaling state of rhodopsin. Meta II binds and activates a large number of photoreceptor-specific G protein molecules, transducins (Gt), by catalyzing the exchange of guanosine triphosphate (GTP) for guanosine diphosphate (GDP) on transducin’s a-subunit, Gta. Deactivation of meta II and consequent Gt-mediated signaling starts with binding of the GPCR kinase called GRK1 (or rhodopsin kinase) that catalyzes subsequent phosphorylation of Ser and Thr residues in the C-terminus of rhodopsin. Phosphorylated rhodopsin then is capped by binding to arrestin, which prevents any residual Gt activation by meta II. The complex of phosphorylated rhodopsin–arrestin loses all-trans-retinal and then arrestin, after which the phosphorylated opsin is dephosphorylated by the action of protein phosphatase 2A (PrP2A). All-trans-retinal is transformed, through a series of steps, to 11-cis-retinal, which rebinds to opsin (as shown in Figure 4), thereby continuing rhodopsin signaling. A fraction of meta II loses all-trans-retinal (without phosphorylation) and directly transforms to opsin.

of the rhodopsin–arrestin complex. Arrestin dissociates from rhodopsin as meta II decays and loses all-trans-retinal. Then phosphorylated opsin is dephosphorylated by protein phosphatase 2A (PrP2A). The resulting free opsin is readily regenerated by 11-cis-retinal and continues recycling through the signaling cascade. A fraction of meta II directly dissociates into all-trans-retinal and opsin.

Rhodopsin Interaction with Other Proteins

According to X-ray crystallographic models and atomic force microscopic studies, H-IV–H-V of rhodopsin contact each other in a rhodopsin dimer. The sizes of Gt, GRK1, and

arrestin proteins also favor the hypothesis of their interaction with the rhodopsin dimers. Previously reported models of rhodopsin with these proteins are discussed below. These complexes have yet to be resolved by crystallography.

Rhodopsin–Gt

In its inactive state, Gt is a membrane-associated protein that consists of a, b, and g subunits with one guanosine diphosphate (GDP) noncovalently bound to the a-subunit. Post-translational modification of the a-(myristoylation) and g-(farnesylation) subunits help this protein to associate with the membrane. A model for the rhodopsin–Gt complex has been reported (Figure 8(a)). Spectroscopic, biochemical, and peptide competition experiments reveal that cytoplasmic loops II, III, H-8, and the C-terminal tail of rhodopsin interact with transducin. The interacting sites of transducin are the C-terminal tail, N-terminal helix, the a4–b6–a5 region of the a-subunit, and the farnesylated C-terminal region of the g-subunit.

Rhodopsin–GRK1

GRK1 phosphorylates multiple sites on the C-terminal tail that is freely accessible in both active and inactive states of rhodopsin (Figure 8(b)). A single activated rhodopsin (meta II) molecule can induce the phosphorylation of hundreds of other rhodopsins. Cytoplasmic loops II and III of rhodopsin are the most important sites for binding of GRK1, and the N-terminal 30 residues of GRK1 are important for this interaction. Inactivating mutations in GRK1 are found in human patients with Oguchi disease, a stationary form of night blindness characterized by a substantial delay in recovery of dark vision after photobleaching.

Rhodopsin–Arrestin

Arrestin binds to photoactivated-phosphorylated rhodopsin (Figure 8(c)). Biochemical analysis of the arrestin–rhodopsin complex reveals that several domains of arrestin are essential for this interaction. In particular, the region from residues 163 to 189 is essential for binding to activated-phosphorylated rhodopsin, but not to unphosphorylated rhodopsin. Lysine and arginine residues of arrestin are also very important for specific binding, but only to phosphorylated rhodopsin.

Mutations in Rhodopsin and Retinal

Diseases

Mutations in the genes encoding many proteins involved in phototransduction and the visual and signaling cycles have been implicated in causing blinding diseases of humans such as Leber’s congenital amaurosis (LCA), Stargardt

Gi-protein

(a)Rhodopsin dimer

GRK1

(b)

Arrestin

(c)

Figure 8 Conceptual models of rhodopsin dimers interacting with Gt, GRK1 and arrestin. (a) Rhodopsin dimer bound to one heterotrimeric Gt. Gta is colored red, Gtb is colored green, and Gtg is colored blue. Gt occupies a single rhodopsin dimer, with only one rhodopsin monomer requiring activation. Helices of rhodopsin are colored as in Figure 2. (b) A rhodopsin monomer is modeled such that its third cytoplasmic loop (C-III) lies close to the proposed receptor-docking site for GRK1. This allows the GRK1 active site to have easy access to the C-tail of activated rhodopsin or of a neighboring unactivated rhodopsin in the same membrane plane, thereby allowing high gain phosphorylation of the ROS. (c) This theoretical model reflects the interaction of one arrestin molecule with a rhodopsin dimer. Molecules are represented in a space-filled background and the plane of the lipid bilayer is shown. No structural optimization was performed.

macular degeneration, congenital cone–rod dystrophy, and retinitis pigmentosa (RP). More than 100 rhodopsin mutants resulting in human eye diseases have been identified (Figure 2). Some mutations result in degeneration of

a-waves

0

00

μV

00

–600

WT

Lrat–/–multigavage Lrat–/–

(d)Intensity (log (cd·s·m2·10))

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rod cells, while some affect the function of rhodopsin. Mutations at the C-terminal tail impair rhodopsin trafficking from RISs to the ROSs. Mutations, for example Pro23 to His, which lead to rhodopsin misfolding will not allow the protein to reach disk membranes of ROS. Nonetheless, the ROSs degenerate and finally cause blindness. The Lys296 mutant is unable to bind chromophore, thereby compromising rhodopsin function. Mutations, for example Ala292 to Glu, which lead to human congenital night blindness do not involve ROS degeneration but rather compromise human vision under dim light. Mutations of proteins in the visual cycle also cause eye diseases. For example, inactivating mutations in the LRAT gene cause LCA. The Lrat–/– knock-out mouse with LRAT-mediated retinal dystrophy evidences only traces of retinoid compounds in ocular tissues, resulting in impaired vision from birth. The ROS are shortened in Lrat / mice, and photoreceptors degenerate very slowly. This disease can be treated by dietary intake of active chromophores or their 9-cis- precursors. Oral supplementation of Lrat / mice with 9-cis-retinyl acetate restored retinal function (Figure 9).

See also: Phototransduction: Phototransduction in Rods; Phototransduction: The Visual Cycle; Rod and Cone

Photoreceptor Cells: Inner and Outer Segments; Rod Photoreceptor Cells: Soma and Synapse.

Further Reading

Arshavsky, V. Y., Lamb, T. D., and Pugh, E. N., Jr. (2002).

G proteins and phototransduction. Annual Review of Physiology 64: 153–187.

Filipek, S., Stenkamp, R. E., Teller, D. C., and Palczewski, K. (2003). G protein-coupled receptor rhodopsin: A prospectus. Annual Review of Physiology 65: 851–879.

Fotiadis, D., Liang, Y., Filipek, S., et al. (2003). Atomic-force microscopy: Rhodopsin dimers in native disc membranes. Nature 421: 127–128.

Hargrave, P. A., McDowell, J. H., Curtis, D. R., et al. (1983). The structure of bovine rhodopsin. Biophysics of Structure and Mechanism 9: 235–244.

Menon, S. T., Han, M., and Sakmar, T. P. (2001). Rhodopsin: Structural basis of molecular physiology. Physiological Reviews 81: 1659–1688.

Muller, D. J., Wu, N., and Palczewski, K. (2008). Vertebrate membrane proteins: Structure, function, and insights from biophysical approaches. Pharmacological Reviews 60: 43–78.

Okada, T., Sugihara, M., Bondar, A. N., et al. (2004). The retinal conformation and its environment in rhodopsin in light of a new 2.2 A˚ crystal structure. Journal of Molecular Biology 342: 571–583.

Palczewski, K. (2006). G protein-coupled receptor rhodopsin. Annual Review of Biochemistry 75: 743–767.

Palczewski, K., Kumasaka, T., Hori, T., et al. (2000). Crystal structure of rhodopsin: A G protein-coupled receptor. Science 289: 739–745.

Park, J. H., Scheerer, P., Hofmann, K. P., Choe, H. W., and Ernst, O. P. (2008). Crystal structure of the ligand-free G-protein-coupled receptor opsin. Nature 454: 183–187.

Park, P. S., Lodowski, D. T., and Palczewski, K. (2008). Activation of G-protein-coupled receptors: Beyond two-state models and tertiary conformational changes. Annual Review of Pharmacology and Toxicology 48: 107–141.

Rao, V. R. and Oprian, D. D. (1996). Activating mutations of rhodopsin and other G protein-coupled receptors. Annual Review of Biophysics and Biomolecular Structure 25: 287–314.

Ridge, K. D. and Palczewski, K. (2007). Visual rhodopsin sees the light: Structure and mechanism of G protein signaling. Journal of Biological Chemistry 282: 9297–9301.

Salom, D., Lodowski, D. T., Stenkamp, R. E., et al. (2006). Crystal structure of a photoactivated deprotonated intermediate of rhodopsin. Proceedings of the National Academy of Sciences of the United States of America 103: 16123–16128.

Travis, G. H., Golczak, M., Moise, A. R., and Palczewski, K. (2007). Diseases caused by defects in the visual cycle: Retinoids as potential therapeutic agents. Annual Review of Pharmacology and Toxicology

47: 469–512.

Glossary

Lipofuscin – Fluorescent pigment granules found within cells of the RPE. Lipofuscin contains oxidized fatty acids and condensation of products of retinaldehyde with phosphatidylethanolamine, such as A2E. Lipofuscin is thought to arise from the incomplete digestion of phagocytosed outer segments. Components of lipofuscin, including A2E, are cytotoxic and thought to play a role in the etiology of macular degeneration.

Opsin visual pigment – Opsin pigments are light-sensitive complexes containing a protein and an 11-cis-retinaldehyde chromophore.

Outer segment – An elongated light-sensitive structure attached to the connecting cilium of rod and cone photoreceptors. The outer segment comprises a stack of approximately 1000 membranous disks. These disks are loaded with rhodopsin or cone opsin visual pigments.

Retinyl ester – A conjugate of vitamin A (retinol) with a fatty acid. Retinyl esters represent stable, nontoxic, and water-insoluble storage forms of retinol. Retinyl esters are also the substrate for Rpe65-isomerase in RPE cells.

Retinaldehyde – An oxidized form of retinol. Retinaldehydes are highly reactive and potentially cytotoxic. The 11-cis isomer of retinaldehyde (11-cis- RAL) is the light-sensitive chromophore in rhodopsin and cone-opsin visual pigments.

Schiff base – It is also called an imine. Results from the reaction of a primary amine (as in lysine or phosphatidylethanolamine) with a carbonyl group (as in retinaldehyde) to form a carbon–nitrogen double bond with loss of a water molecule. Formation of a Schiff base is reversible.

The vertebrate retina contains two classes of light-sensitive cells, rods and cones. Both cell types contain a membranous structure called the outer segment (OS), which are loaded with rhodopsin or cone-opsin visual pigments. These pigments are members of the G-protein-coupled receptor superfamily. Each rod OS contains approximately 108 rhodopsin pigments. The ligand for these pigments is 11-cis-retinaldehyde (11-cis-RAL), which is covalently coupled to a lysine in the opsin protein through a Schiff-base linkage. Absorption of a photon

by an opsin pigment induces photoisomerization of the 11-cis-RAL chromophore to all-trans-retinaldehyde (all-trans-RAL). This isomerization converts the pigment to active metarhodopsin II, which stimulates the visualtransduction cascade. After a brief period, metarhodopsin II is inactivated by rhodopsin kinase-mediated phosphorylation and subsequent capping by arrestin. Next, all- trans-RAL dissociates from the inactivated opsin pigment. To restore light sensitivity, the bleached apo-opsin recombines with another 11-cis-RAL, forming a new rhodopsin or cone-opsin pigment. To maintain continuous vision in light, the all-trans-RAL released by bleached pigments must be converted back to 11-cis-RAL. This process is carried out by a multistep enzyme pathway called the visual cycle (Figure 1). The first two catalytic steps of this pathway occur in photoreceptors, while the remaining steps take place in cells of the retinal pigment epithelium (RPE). The RPE is an epithelial monolayer adjacent to the photoreceptors. Apical processes of RPE cells interdigitate with the photoreceptor OS. The regeneration of visual chromophore is one of the several collaborations between photoreceptors and RPEs; and cone opsins may have access to an alternative source of 11-cis-RAL chromophore. This alternative retinoid pathway is present in Mu¨ller glial cells.

Clearance of All-trans-RAL from OS Disks

Following photoactivation and subsequent deactivation of the opsin pigment, all-trans-RAL probably exits between transmembrane (TM) helices, TM1 and TM7, into the lipid bilayer. The all-trans-RAL diffuses within the bilayer until it encounters the amine headgroup of a phosphatidylethanolamine, which may condense with the all-trans-RAL to form the Schiff base, N-retinylidene-phosphatidylethanol- amine (N-ret-PE). This condensation reaction is reversible. On the cytoplasmic surface of the OS disk-membrane, all- trans-RAL is reduced to all-trans-retinol (all-trans-ROL), driving dissociation of N-ret-PE (see below). However, all- trans-RAL can be temporarily trapped as N-ret-PE on the intradiscal surface. An adenosine triphosphate (ATP)- binding cassette transporter called ABCA4 (also ABCR or rim-protein) is present in the disks of rod and cone OS. Mice with a knockout mutation in the abca4 gene show delayed clearance of all-trans-RAL and elevated N-ret-PE in the retina following exposure to light. In vitro studies suggest that ABCA4 is an outwardly directed flippase for

the removal of all-trans-RAL from disk membranes for subsequent reduction to all-trans-ROL. Mutations in the human ABCA4 gene cause Stargardt macular degeneration and a subset of recessive cone–rod dystrophy in humans. Stargardt patients and abca4 –/– mice accumulate toxic lipofuscin pigments in RPE cells. Buildup of these fluorescent pigments is important in the pathogenesis of photoreceptor degeneration in Stargardt’s disease.

Reduction of All-trans-RAL to

All-trans-ROL

This reaction is carried out in photoreceptor OS by a member of the short-chain dehydrogenase/reductase family called photoreceptor retinol dehydrogenase (prRDH) or RDH8. RDH8 uses nicotinamide adenine dinucleotide phosphate oxidase (NADPH) as a co-factor. In rdh8 –/– knockout mice, reduction of all-trans-RAL to all-trans- ROL is slowed but not halted, suggesting that RDH8 function is complemented in photoreceptors by at least one other retinol dehydrogenase. Photoreceptors contain a second retinol dehydrogenase called RDH12 that also catalyzes NADPH-dependent reduction of all-trans-RAL to all-trans-ROL. Mice with a knockout mutation in the rdh12 gene show mildly slowed reduction of all-trans-RAL to all-trans-ROL, and protection from light-induced

photoreceptor degeneration. Unlike RDH8, which is expressed in photoreceptor OS, RDH12 is expressed in photoreceptor inner segments. This distribution is unexpected given that all-trans-RAL is released following light exposure into the OS. RDH12 may play a detoxifying role in the inner segment by reducing all-trans-RAL that escaped reduction by RDH8 in the OS. Mutations in RDH12 cause a severe recessive blinding disease called Leber congenital amaurosis (LCA). No mutations in RDH8 have been associated with a retinal dystrophy in humans. Mice with a knockout mutation in the rdh8 gene show normal kinetics of rhodopsin regeneration and delayed recovery of sensitivity following exposure to bright light. An identical pattern is seen in abca4 –/– mice. ABCR and all-trans- ROL dehydrogenase act sequentially in the visual cycle to remove all-trans-RAL following a photobleach (Figure 1). Delayed dark adaptation in rdh8 –/– and abca4 –/– mice is probably due to noncovalent reassociation of all-trans- RAL with apo-opsin to form a noisy photoproduct that activates transducin.

Transfer of All-trans-ROL from

Photoreceptors to the RPE

Interphotoreceptor retinoid-binding protein (IRBP) is secreted by photoreceptors and present at a high concentration in the extracellular space. Besides IRBP, this space is filled with extracellular matrix material and is called the

interphotoreceptor matrix (IPM). IRBP contains binding sites for both 11-cis- and all-trans-retinoids. IRBP has been shown to accelerate the removal of all-trans-ROL from bleached photoreceptors. The uptake of all-trans- ROL by IRBP may involve a receptor on the OS plasma membrane. Retinoids bound to IRBP are protected from oxidation and isomerization during transit through the IPM. Mice with a knockout mutation in the irbp gene show accumulation of all-trans-ROL in the retina, and reduced all-trans-REs in the RPE following light exposure. These mice also show accumulation of 11-cis-RAL in the RPE and reduced 11-cis-RAL in the retina following light exposure. These results suggest that IRBP functions to extract all-trans-ROL from bleached photoreceptors, and 11-cis-RAL from RPE cells. Mutations in the RBP3 gene for IRBP cause the inherited blinding disease, recessive retinitis pigmentosa in a small subset of cases.

Another all-trans-ROL-binding protein, cellular retinol– binding protein type-1 (CRBP1), is present in RPE cells. CRBP1 is a soluble protein that binds all-trans-ROL with 100-fold higher affinity than does IRBP. This difference in affinity drives the uptake of all-trans-ROL from the IPM into RPE cells. Compared with wild-type mice, crbp1 –/– knockout mice contain reduced all-trans-REs in the RPE and higher all-trans-ROL in the retina following light exposure. This biochemical phenotype is similar to the phenotype in irbp–/– mice.

Synthesis of Retinyl Esters

The major retinyl-ester synthase in RPE cells is lecithin: retinol acyl transferase (LRAT), which catalyzes the transfer of a fatty-acyl group from the sn1 position in phosphatidylcholine to all-trans-ROL (see Figure 1). The resulting all-trans-retinyl esters (all-trans-REs) are water-insoluble, and represent a stable and nontoxic storage form of vitamin A. Mice with a knockout mutation in the lrat gene contain virtually no all-trans-REs or other visual retinoids in their ocular tissues. Accordingly, lrat–/– mice are totally blind. Mutations in the human LRAT gene are yet another cause of recessive LCA.

Another retinyl-ester synthase activity, called acyl-CoA: retinol acyltransferase (ARAT), is present in RPE cells. Unlike LRAT, ARAT uses palmitoyl coenzyme A (palm CoA) as an acyl donor. Two enzymes have been shown to posses ARAT activity. Diacylglycerol acyltransferase type-1 (DGAT1), which catalyzes palm CoA-dependent synthesis of triglycerides from diacylglycerol, also catalyzes palm CoA-dependent synthesis of all-trans-REs from all- trans-ROL. Multifunctional O-acyltransferase (MFAT) also

possesses ARAT catalytic activity. The very low level of all-trans-REs in the RPE of lrat–/– mice despite the pres-

ence of ARAT activity is due to the 10-fold higher KM for all-trans-ROL substrate of ARAT versus LRAT. ARAT

preferentially uses free all-trans-ROL as a substrate in contrast to LRAT, which uses holo-CRBP1.

Retinoid Isomerization

Conversion of a planar all-trans-retinoid to the strained 11cis configuration is energetically unfavorable. Rpe65-isom- erase uses all-trans-REs as substrate and catalyzes two reactions: hydrolysis of the carboxylate ester, and trans to cis isomerization of the C11–C12 double bond in the retinoid. Accordingly, the energy released by ester hydrolysis (–5.0 kcal mole–1) is used to drive isomerization (þ4.1 kcal mole–1). Rpe65 is homologous to b-carotene oxygenase in mammals and apocarotene oxygenase (ACO) in cyanobacteria. The X-ray diffraction analysis showed that ACO has a seven-bladed b-propeller structure, with a Fe2+-4-His arrangement at its axis. The four His residues that define the Fe2+-binding site are conserved in all members of the ACO family including Rpe65. Rpe65 was shown to bind Fe2+, which is required for its catalytic activity. Rpe65 is strongly associated with membranes but contains no membrane-spanning segments. Mice with a knockout mutation in the rpe65 gene contain high levels of all-trans-REs in the RPE and no detectable 11-cis-RAL. Accordingly, rpe65 –/– photoreceptors contain only apoopsin, and the mice have no detectable visual function. Despite blocked synthesis of visual chromophore, photoreceptor morphology is nearly normal in rpe65 –/– mice. Visual function has been restored in rpe65 –/– mice and dogs by administering exogenous visual chromophore. Injection of recombinant adeno-associated virus (AAV) containing a wild-type rpe65 gene into the subretinal space (between RPE cells and photoreceptors) of rpe65 –/– mice partially rescued the blindness phenotype. More recently, patients with RPE65-mediated LCA received subretinal injections of a similar RPE65-containing AAV. Encouragingly, these blind patients partially recovered visual function with expression of wild-type Rpe65 in their RPE.

The 11-cis-ROL synthesized by Rpe65 binds to cellular retinaldehyde-binding protein (CRALBP) in RPE cells. CRALBP also binds 11-cis-RAL. Mutations in the gene for CRALBP (RLBP1) cause several inherited retinal dystrophies including recessive retinitis pigmentosa. A newly synthesized molecule of 11-cis-ROL has two potential fates. As discussed below, it can be oxidized to 11-cis-RAL for use as visual chromophore. Alternatively, it can be esterified by LRAT to form an 11-cis-RE. 11-cis-REs represent a storage form of preisomerized chromophore precursor. Hydrolysis of 11-cis-REs is catalyzed by 11-cis-retinyl ester hydrolase (11-cis-REH) in the plasma membrane of RPE cells. The protein responsible for 11-cis-REH activity in RPE cells has not yet been identified.

Synthesis of 11-cis-RAL Chromophore

The final step in the visual cycle is oxidation of 11-cis-ROL to 11-cis-RAL. This reaction is catalyzed by 11-cis-ROL- dehydrogenase type-5 (RDH5), which uses NAD+ as a cofactor. Mice with a knockout mutation in the rdh5 gene show accumulation of 11-cis-ROL and 11-cis-REs in the RPE, and delayed recovery of rod sensitivity following light exposure. 11-cis-RAL is synthesized inrdh5 –/– mice, albeit at a reduced rate, suggesting that RPE cells express at least one other 11-cis-ROL-dehydrogenase. RDH11 catalyzes NADP+-dependent oxidation of 11-cis-ROL to 11-cis-RAL in the RPE. Surprisingly,rdh5 –/–, rdh11 –/– double-knockout mice also synthesize 11-cis-RAL, although more slowly than in rdh5 –/– or rdh11 –/– single-knockout mice, and much more slowly than in wild-type mice. Thus, extensive functional redundancy exists for the oxidation of 11-cis-ROL in RPE cells, similar to the functional redundancy for reduction of all-trans-RAL in photoreceptors. 11-cis-RAL is strongly bound to CRALBP in RPE cells. CRALBP has been shown to interact with a protein complex on the cytoplasmic surface of the apical plasma membrane. From this position, 11-cis- RAL is transferred across the plasma membrane to bind IRBP in the IPM. This process may involve a receptor for IRBP on RPE cells.

Regeneration of Rhodopsin or

Cone Opsin

The final step in the visual cycle is regeneration of a visual pigment from an apo-opsin and 11-cis-RAL. The mechanism whereby 11-cis-RAL is transferred from IRBP in the IPM to apo-opsin in the OS disk is unknown. It may involve an IRBP receptor on the OS plasma membrane, or simple diffusion of the 11-cis-RAL. No retinoid-binding protein has been identified in OS. The interaction of 11-cis-RAL with an apo-opsin involves a two-step process. First, a weak noncovalent complex is formed with 11-cis- RAL binding to a hypothesized entrance site on the opsin. Second, the 11-cis-RAL moves into the hydrophobic pocket and forms a Schiff base. This step is virtually irreversible in the case of rhodopsin. Once formed, rhodopsin is extremely quiet, with a spontaneous thermalactivation rate of one isomerization every 2000 years. In contrast to rhodopsin, recombination of 11-cis-RAL with the apo-cone-opsins is less favorable thermodynamically. Unlike rhodopsin, 11-cis-RAL freely dissociates from coneopsins. For example, a dark-adapted red cone contains approximately 10% apo-cone-opsin due to spontaneous dissociation of chromophore. This effect contributes to the higher noise and much lower sensitivity of cones versus rods. It is also explains the tendency of rods to steal visual chromophore from cones when the availability of 11-cis- RAL is limited.

Regulation of the Visual Cycle

In the dark, photoreceptors stop releasing all-trans-ROL. Residual all-trans-ROL is esterified by LRAT. The major retinoids present in a dark-adapted eye are all-trans-REs in the RPE and 11-cis-RALs in photoreceptor visual pigments. How does the visual cycle know to stop converting all-trans-REs into 11-cis-RAL chromophore in the dark? One mechanism is the strong inhibition of Rpe65 by its product, 11-cis-ROL. When rhodopsin is fully regenerated and CRALBP is saturated, further synthesis of 11-cis-ROL by Rpe65 is inhibited.

A second mode of visual-cycle regulation involves an opsin protein called RPE-retinal G-protein receptor (RGR) opsin, expressed in RPE cells. Within RPE cells, all- trans-REs are stored in two compartments, internal membranes and oil droplets. Rpe65 associates with internal membrane but not lipid droplets. Hence, RPE internal membranes contain a pool of all-trans-REs available as substrate for isomerization, while lipid droplets contain a storage pool of all-trans-REs. This storage pool is potentially much larger than the isomerase pool in membranes. RGR opsin mediates light-dependent transfer of all-trans- REs from the storage compartment to the membrane compartment for isomerization. In light, where the requirement for visual chromophore is high, RGR opsin stimulates synthesis of 11-cis-ROL by increasing substrate availability to Rpe65. Consistently, mice with a knockout mutation in the rgr gene synthesize less 11-cis-RAL in the light and accumulate all-trans-REs. Mutations in the human RGR gene cause autosomal dominant retinitis pigmentosa.

See also: Phototransduction: Inactivation in Cones; Phototransduction: Inactivation in Rods; Phototransduction: Phototransduction in Cones; Phototransduction: Phototransduction in Rods; Phototransduction: Rhodopsin.

Further Reading

Batten, M. L., Imanishi, Y., Maeda, T., et al. (2004). Lecithin–retinol acyltransferase is essential for accumulation of all-trans-retinyl esters in the eye and in the liver. Journal of Biological Chemistry 279: 10422–10432.

Beharry, S., Zhong, M., and Molday, R. S. (2004). N-retinylidene- phosphatidylethanolamine is the preferred retinoid substrate for the photoreceptor-specific ABC transporter ABCA4 (ABCR). Journal of Biological Chemistry 279: 53972–53979.

Cideciyan, A. V., Aleman, T. S., Boye, S. L., et al. (2008). Human gene therapy for rpe65 isomerase deficiency activates the retinoid cycle of vision but with slow rod kinetics. Proceedings of the National Academy of Sciences of the United States of America 105: 15112–15117.

Gollapalli, D. R. and Rando, R. R. (2003). All-trans-retinyl esters are the substrates for isomerization in the vertebrate visual cycle. Biochemistry 42: 5809–5818.

Jin, M., Li, S., Moghrabi, W. N., Sun, H., and Travis, G. H. (2005). Rpe65 is the retinoid isomerase in bovine retinal pigment epithelium. Cell 122: 449–459.

Kaschula, C. H., Jin, M. H., Desmond-Smith, N. S., and Travis, G. H. (2006). Acyl coa:retinol acyltransferase (ARAT) activity is present in bovine retinal pigment epithelium. Experimental Eye Research 82: 111–121.

Kefalov, V. J., Estevez, M. E., Kono, M., et al. (2005). Breaking the covalent bond – a pigment property that contributes to desensitization in cones. Neuron 46: 879–890.

Lamb, T. D. and Pugh, E. N. (2004). Dark adaptation and the retinoid cycle of vision. Progress in Retinal and Eye Research

23: 307–380.

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Glossary

Blood flow autoregulation – The property of a tissue or an organ (i.e., retina) to maintain constant blood flow during changes in perfusion pressure. Blood–retinal barrier – The system of occluding cellular junctions in the retinal pigmented epithelium and retinal vascular endothelium that prevents free movement of fluid and macromolecules from the blood into the retina.

Cilioretinal artery – A retinal artery which arises from the choriod or the posterior ciliary artery, and is found as a variant in some individuals.

Cotton wool spots – White patches on the retina observed on fundus examination and are caused by local obstruction of the tiny arteries supplying that area.

Fluorescein fundus angiography – Fundus photographs taken in rapid sequence following injection of the fluorescent dye, fluorescein. This provides important information about blood flow as the dye reaches the retinal and choroidal vasculature.

The retina has a dual blood supply; the retinal vasculature supplies only the inner retinal layers up to the inner part of the inner nuclear layer (Figure 1), while the choroidal vascular bed supplies the outer 130 mm – up to the outer part of the inner nuclear layer, so that retinal vessels supply only 20% of the retina while the choroid supplies 80%. In this article, the discussion is restricted to the retinal component.

Arterial Supply of the Retina

The main arterial supply of the retina is by the central retinal artery (CRA). In some eyes, another artery, called the cilioretinal artery, may supply a highly variable part of the retina.

Central Retinal Artery

The CRA is usually the first branch of the ophthalmic artery, arising as an independent branch or in common

with one of the posterior ciliary arteries (Figure 2). Its course can be divided into three distinct parts: (1) intraorbital (lying below the optic nerve (ON) – (Figure 2)),

(2) intravaginal (lying in the space between the ON and its sheath), and (3) intraneural (lying in the ON) (Figure 3). It enters the ON about 10 mm posterior to the eyeball (Figures 2 and 3). A variable number of branches arise from each of its three parts, which anastomose with the surrounding branches from other arteries, mostly in the pial plexus of the ON (Figure 3). At the optic disk, the CRA usually first divides into two and then each of them further divides into its various branches (Figure 4). The lumen of the intraneural part of the CRA is approximately 200 mm.

Cilioretinal Artery

The cilioretinal artery is either a direct branch of one of the posterior ciliary arteries or arises from the peripapillary choroid and enters the retina by hooking around the Bruch’s membrane at the disk margin – usually on the temporal side (Figures 3 and 5). Based on ophthalmoscopy, the incidence of the occurrence of the cilioretinal artery reported by different authors varies from 6% to 25%. However, fluorescein fundus angiography provides the most reliable data because the cilioretinal artery fills synchronously with the choroidal filling, which usually starts to fill before the retinal circulation (Figure 6(a)). An artery which, on ophthalmoscopy, may look similar to a cilioretinal artery may in fact be an intraneural branch of the CRA emerging at the optic disk – not a true cilioretinal artery. A fluorescein fundus angiographic study of 2000 eyes showed one or more cilioretinal arteries in 32% of the eyes and in both eyes in 15% of persons. There is great variability in size, number, and distribution of the cilioretinal arteries. The area of the retina supplied by the cilioretinal arteries varies markedly, from a tiny region to a large sector of the retina. Eyes where one-fourth to half of the retina is supplied by a cilioretinal artery have been observed (Figure 6(a)). Rarely, the CRA is missing and the entire retina is supplied by the cilioretinal artery (Figure 6(b)). The outer part of the entire retina is always supplied by the posterior ciliary artery. When a cilioretinal artery is present, in the part of the retina supplied by it, the entire thickness of the retina receives its blood supply from the posterior ciliary artery.

The blood flow in the retinal vascular bed depends upon the perfusion pressure, which is equal to the difference