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

subunits are soluble, the holo-transducin is firmly anchored to the disk membranes by a farnesyl lipid group that is post-translationally attached to the carboxy-terminal of the g-subunit and an acyl group on the amino-terminal of the a-subunit. Therefore, photo-activated rhodopsin (R*) can only interact and activate transducin through lateral diffusion at the membrane surface.

Like many other G proteins, transducin exists in one of the two states: the GDP-bound inactive state and the GTPbound active state. Binding of R* catalyzes the exchange of GTP for GDP on the a-subunit. The active Ga-GTP (G*) dissociates from R* as well as its native partner, Gbg, and interacts with PDE to carry the signal forward. In the meantime, R* is able to activate additional molecules of transducin. Transducin activation by R* represents the first amplification step in the phototransduction cascade. The estimated rate of transducin activation by a single R* varied from 10 to over 3000 s 1 at room temperature. A rate of 120 s 1 was later reported to be more consistent with biochemical, light-scattering, and electrophysiological measurements. The rate is roughly doubled in mammalian rods due to the higher body temperature. Until recently, it was believed that over a hundred transducins are activated during the lifetime of a single R* in mammalian rods. This number is now revised to be 20 in mouse rods, based on the shorter life time of R* (80 ms) and activation rate of transducin by R* (240 s 1).

The importance of transducin for conveying the signal from R* to PDE was manifested from animal models deficient in Ga and human patients carrying Ga mutations. It was found that rods of Gat1-null mice (Gnat1–/–) lost all light sensitivity. In human, a mis-sense mutation in Gnat1 (encoding the rod Gat1) is implicated in autosomal-dominant congenital stationary night blindness of Nougaret, caused by constitutive activation of rod phototransduction. The gnat1–/– mouse line has proven to be a valuable tool for blocking rod phototransduction to study cone phototransduction and circadian photoreception. It was also used successfully to delineate two apoptotic pathways in light-induced retinal degeneration. Bright light triggers apoptosis of photoreceptors through a mechanism requiring the activation of rhodopsin but not transducin signaling. In contrast, low-intensity light induces apoptosis that is predominantly dependent on transducin signaling.

The High Catalytic Power of PDE

Accounts For the Second Amplification

Step

PDE is the third component of rod phototransduction. It is a hetero-tetrameric protein consisting of two catalytic subunits, a- and b-, and two identical g-subunits. PDE is anchored to the disk membrane by a hydrophobic

isoprenyl group (compounds that are derived from isoprene, 2-methylbuta-1,3-diene, linearly linked together) posttranslationally attached to the C-termini of the two catalytic subunits. As for transducin activation, PDE activation by G is through lateral diffusion on the rod disk membrane. Each catalytic subunit has two high-affinity noncatalytic binding sites and one catalytic binding site for cGMP. The noncatalytic sites were suggested to modulate the binding affinity between PDEg and PDEab. The amount of PDE is ~1–2% of rhodopsin. Thus, the first three components of phototransduction are present in the ratio of 100R:10G:1PDE. In the dark, the two g-subunits act as inhibitory subunits by binding to the two catalytic subunits and significantly reducing the hydrolysis of cGMP. In the light, Ga-GTP encounters PDEg and sterically displaces the latter, therefore relieving its inhibitory effect on the catalytic subunits and permitting the hydrolysis of cGMP to proceed (Figure 2). Since each G* can only activate one PDEg, two G*’s are required to fully activate a holo PDE. This is likely the scenario in vivo during light activation due to the excess amount of G over PDE and the presence of many molecules of G* activated by rhodopsin.

In contrast to the amplification achieved during transducin activation by R*, the activation of PDE by G* constitutes no gain, that is, with an efficiency approaching 1 (one G*, one activated PDE catalytic subunit) or 0.5 in terms of PDE holoenzyme. It is the catalytic power of PDE* that provides the second amplification step. It was reported that PDE* hydrolyzes cGMP at a rate close to the limit set by aqueous diffusion, with a Km of 10 mM

and a Kcat of 2200 s 1, making it one of the most efficient enzymes in vivo.

In addition to the noise produced by spontaneous activation of rhodopsin, spontaneous activation of individual catalytic PDE subunits produces the continuous noise, which accounts 30–80% (depending on the species) of the total dark noise variance in rods. The basal spontaneous PDE activity balances constitutive guanylate cyclase activity in the dark, therefore maintaining a steady-free cGMP level. It also has the function of increasing the rate of cGMP turnover and consequently speeding up the dim flash response.

One might have expected that the deletion of PDEg from mouse rods would unleash the full catalytic power of PDEab. However, it was found, in the absence of PDEg that the PDEab dimer actually lacked catalytic activity, and the photoreceptors of the mutant mouse rapidly degenerated. Thus, the inhibitory PDEg subunit appears to be necessary for the integrity of the catalytic PDEab subunits. The degeneration might be caused by an abnormally high cGMP concentration due to the lack of hydrolysis. A related example is the rd mouse, which is the oldest and one of the best-known models for retinal degeneration. The rod cells in the rd mouse begin to degenerate at about postnatal day 8, followed by cones;

by 4 weeks, virtually no rod photoreceptors are left. Degeneration in this mouse model is preceded by the accumulation of cGMP in the retina, correlated with deficient activity of the rod PDE due to a mutation in the PDEb subunit. It is worth noting that the rd mouse was instrumental in suggesting that inner retinal neurons could mediate non-image-forming vision.

cGMP Is the Second Messenger Mediating

Rod Phototransduction

By 1970, scientists generally believed that a second messenger was required to mediate the rod photoresponse based on several lines of evidence. First, light absorption occurs on the rod disk membrane, whereas the light-sensitive conductance is in the plasma membrane. Since rod disks are separate from the plasma membrane, a second messenger is required to connect the two. Second, the dim-flash response of rods lasts a few seconds, which is too long to be accounted by the open time of known membrane conductance. However, it took more than a decade before the identity of the second messenger was finally determined to be cGMP. The fierce battle was fought on the validity between two competing candidates, Ca2þ and cGMP. According to the Ca2þ hypothesis, which was first proposed by Hagins, the concentration of intracellular free Ca2þ is low in the dark and rises in the light to block light-sensitive current. The main supporting evidence is that reducing the concentration of external Ca2þ dramatically increases the dark current, suggesting that internal Ca2þ inhibits the dark current. On the other hand, the cGMP hypothesis proposed that the concentration of cGMP was high in the dark to maintain a cGMP-dependent conductance. Light led to the hydrolysis of cGMP and the subsequent closing of the conductance. The supporting evidence is that intracellular injection of cGMP increases the amplititude and latency of the photoresponse. Adding to the complexity is the finding that the free cGMP concentration varies inversely with the free Ca2þ concentration in rods, making it difficult to separate the effect of the two.

This debate was finally settled with the discovery of cGMP-gated channels in rods by Fesenko and colleagues in 1985. By using the patch-clamp technique, they showed that cGMP increased a cation conductance of inside-out patches of outer-segment plasma membrane without the need of ATP. The direct channel gating by cGMP is surprising because cyclic nucleotides were generally believed to act through cyclic-nucleotide-dependent kinases and protein phosphorylation on target proteins at that time. This dogma partially explained scientists’ reluctance to embrace the cGMP hypothesis because protein phosphorylation was too slow. Another monumental work by Yau and Nakatani was published at the same year that helped the anointment of cGMP as the

right candidate. An identical cGMP-gated cation conductance was found on a truncated rod outer segment with an intact plasma membrane. Most importantly, this conductance could be suppressed by light, suggesting that the long-sought light-sensitive conductance is the cGMPgated conductance. The publications by Fesenko and Yau marked the end of the Ca2þ hypothesis.

The cGMP-Gated Channel Provides the

Final Step of Signal Amplification

The cGMP-gated channel belongs to the family of cyclic- nucleotide-gated (CNG) channels, which are nonselective cation channels. The channel is located on the plasma membrane with a density of 400–1000 mm 2 and is the last component in the activation phase of phototransduction. Rod CNG channels consist of CNGA1 (or a1) and CNGB1 (or b1) subunits. CNGA1 subunit forms functional homomeric channels by themselves when heterologously expressed. Although CNGB1 does not form functional channels by themselves, it confers several properties typical of native channels when coexpressed with the CNGA1 subunit: flickery opening behavior, increased sensitivity to L-cis-diltiazem, (a CNG channel– specific inhibitor) and weaker block by extracellular calcium. For a long time, the rod channel was believed to be a hetero-tetramer consisting of two CNGA1 and two CNGB1 subunits. In 2002, a number of laboratories made the surprising discovery that the rod channel actually has a 3CNGA1:1CNGB1 subunit composition. In humans, mutations in CNGA1 cause retinitis pigmentosa. CNGB1 subunits were found to be crucial for the targeting of the native CNG channel in rods. Thus, only trace amounts of the CNGA1 subunit were found on the rod outer segments in CNGB1-null mice and the majority of rod photoreceptors failed to respond to light.

The gating of the rod channel by cGMP is cooperative with a Hill coefficient of 3; therefore, the light-triggered suppression of the dark current is 3 times larger than the decrease in the intracellular cGMP concentration. This is the last step of signal amplification in rod phototransduction. The combined amplification provided by rhodopsin, PDE, and CNG channels is very high ( 105–106), ensuring the high sensitivity of rods, including the ability of rods to detect single photons.

In the dark, the concentration of free cGMP in the rod outer segment was estimated to be several mM, which is lower than the K1/2 ( 10–40 mM depending on Ca2þ concentration), the concentration of cGMP necessary to half-maximally activate the channel. As a result, only 1% of the CNG channels are open! In other words, 99% of the channels are already closed in the dark and light can only suppress the remaining 1% channels. This explains

why current induced by cGMP injection is more than 10 times larger than the dark current.

The inward current through the cGMP channel is composed of 85% Naþ because Naþ is the predominant external cation and the channel is nonselective to monovalent cations. The remaining current is mainly carried by Ca2þ with a minor contribution from Mg2þ. Extracellular Ca2þ actually partially blocks the channel to reduce its conductance under physiological conditions. The inward current is balanced by an outward current flowing across the inner-segment membrane, which is mainly carried by potassium channels. This circulating current is also called dark current in both rods and cones. Unlike other ligandgated channels, the CNG channel does not desensitize to cGMP, which is important for rods to maintain a steady dark current ranging between 20 and 70 pA in vertebrate rods. The rod photoresponse is essentially a transient suppression of the circulating current. It was estimated that the dark current was carried by 10 000 channels. The participation of large numbers of micro-channels averages out the channels noise, that is, reduces an otherwise substantial stochastic channel noise if the dark current were carried out by a few macro-channels. This feature improves the sensitivity of rods.

Two extrusion mechanisms are critical in maintaining ionic balance in rods. An energy-dependent Na–K ATPase at the inner segment pumped Naþ out and Kþ into the cells. A Na/Ca,K exchanger (NCKX) in the outer-segment plasma membrane extrudes one Ca2þ and one Kþ outward in exchange for four Naþ inward producing the net entry of one positive charge. The exchanger and the CNG channel were found to form a stable complex on the plasma membrane, likely as a way to control the stoichiometry between the two, which is critical for regulating Ca2þ concentration in the rod outer segment. During the light response, the influx of Ca2þ is reduced due to the closure of some CNG channels while

the efflux of Ca2þ through the exchanger is maintained. The resulting Ca2þ decline triggers negative feedback to produce light adaptation.

See also: Phototransduction: Adaptation in Cones; Phototransduction: Adaptation in Rods; Phototransduction: Inactivation in Cones; Phototransduction: Inactivation in Rods; Phototransduction: Phototransduction in Cones; Phototransduction: The Visual Cycle; Primary Photoreceptor Degenerations: Retinitis Pigmentosa; Primary Photoreceptor Degenerations: Terminology; Secondary Photoreceptor Degenerations: AgeRelated Macular Degeneration; Secondary Photoreceptor Degenerations.

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.

Baylor, D. A., Lamb, T. D., and Yau, K. W. (1979a). The membrane current of single rod outer segments. Journal of Physiology 288: 589–611.

Baylor, D. A., Lamb, T. D., and Yau, K. W. (1979b). Responses of retinal rods to single photons. Journal of Physiology 288: 618–634.

Burns, M. E. and Arshavsky, V. Y. (2005). Beyond counting photons: Trials and trends in vertebrate visual transduction. Neuron 48: 387–401.

Burns, M. E. and Baylor, D. A. (2001). Activation, deactivation, and adaptation in vertebrate photoreceptor cells. Annual Review of Neuroscience 24: 779–805.

Fu, Y. and Yau, K. W. (2007). Phototransduction in mouse rods and cones. Pflugers Archiv – European Journal of Physiology 454: 805–819.

Luo, D. G., Xue, T., and Yau, K. W. (2008). How vision begins: An odyssey. Proceedings of the National Academy of Sciences of the United States of America 105: 9855–9862.

Pugh, E. N., Jr. and Lamb, T. D. (2000). Phototransduction in vertebrate rods and cones: Molecular mechanisms of amplification, recovery and light adaptation. In: Stavenga, D. G., de Grip, W. J., and Pugh, E. N., Jr. (eds.) Handbook of Biological Physics, Vol. 3: Molecular Mechanisms of Visual Transduction, pp. 183–255. Amsterdam: Elsevier.

Glossary

GPCRs – G protein-coupled receptors are membrane receptor proteins with a seventransmembrane helical topology that are capable of activating G proteins.

G proteins – Heterotrimeric intracellular proteins so named because they bind to the guanine nucleotides, guanosine diphosphate in an inactive state and guanosine triphosphate in an active state. GRK1 – G protein-coupled receptor kinase 1 (rhodopsin kinase) is a highly specific protein kinase that catalyzes phosphorylation of photoactivated rhodopsin thereby triggering its deactivation.

LCA – Leber’s congenital amaurosis is an inherited degenerative disease of the retina that results in a severe loss of vision.

Photoisomerization – The structural change between chromophore geometric isomers (cis to trans) caused by photoexcitation. For rhodopsin, photoisomerization of its chromophore leads to activation of this receptor.

ROSs – Rod outer segments are the cylindrical outer portions of rod cells, each containing hundreds of membranous disks enveloped by the cellular (plasma) membrane.

RP – Retinitis pigmentosa is the name of a heterogeneous group of progressively blinding degenerations of the human retina caused by mutations in genes encoding photoreceptor proteins with an autosomal dominant (adRP), autosomal recessive (arRP), or X-linked pattern of inheritance. Schiff base – A functional chemical group containing a carbon–nitrogen double bond. The retinylidene moiety is a chemical link between retinal and an amino group, for example, Lys296 of opsin.

Vision is an important biological sensing mechanism that involves conversion of light signals received by the eye into electrical nerve impulses transmitted to the brain by a process called phototransduction, consisting of a cascade of biological processes that occur in photoreceptor cells (rod and cone cells) of the retina. In the absence of light, photoreceptors are depolarized to a membrane resting potential of –40 mV. In the presence of light, the plasma membrane of the photoreceptor cells becomes hyperpolarized to –70 mV, resulting in a reduced amount of

neurotransmitter released to downstream neurons. This article focuses on rhodopsin structure that relates to its function as a G protein-coupled receptor (GPCR).

Rod Cells and Rhodopsin

The vertebrate rod cell, a highly differentiated postmitotic neuron, is characteristically long, cylindrical, and primarily consists of an outer segment connected to an inner segment via a cilium (Figures 1(a) and 1(b)). The rod outer segment (ROS) contains a stack of disk membranes enclosed by the plasma (cell) membrane, whereas the rod inner segment (RIS) contains the metabolic machinery for this cell. Rhodopsin is processed in the endoplasmic reticulum and transferred to the Golgi membranes of the RIS for additional processing of its carbohydrate moieties. Then rhodopsin-containing Golgi vesicles fuse with the apical plasma membrane of the inner segment and the rhodopsin molecules are transported through the rod cell cilium to the ROS where they form disk membranes. Mutations in the C-terminal region of rhodopsin inhibit transport of rhodopsin to ROS, indicating that this region is essential for recognition by the transport machinery.

A mammalian ROS consists of a stack of 1000–2000 disks enclosed by the plasma membrane. A cryo-electron tomography image of murine ROS (Figure 1(c)) also reveals that the thickness of a single disk membrane is 8 nm. Rhodopsin comprises >90% of all proteins in disk membranes and occupies 50% of the disk membrane volume. It is also present at a lower density in the plasma membrane of rod cells, and its expression is essential for ROS formation, which is absent in knock-out Rho–/– mice. In wild-type mice, there are approximately 8 104 rhodopsin molecules per disk and 3.96 1014 per eye. The power spectra of negatively stained disk membranes from bovine ROS (Figure 1(d)) reveals a diffuse diffraction ring at

˚ –1

(45 A) , indicating paracrystallinity of rhodopsin. Organization of the seven helices of rhodopsin was first ascertained by using a low-resolution imaging method called electron crystallography. Rhodopsin is unequally distributed in disk membranes. Electron tomographs of the ROS reveal both highand low-density regions (Figure 1(e)). An atomic force microscopic image of native disk membranes showed that the average packing density of rhodopsin monomers is 48 300 8000 mm–2. Recent atomic force microscopic studies disclosing the arrangement of rhodopsin in native mouse disk membranes revealed that rhodopsin and opsin form structural dimers arranged in

paracrystalline arrays of rows (Figure 1(f )). Previous studies also support the importance of dimerization/oligomerization as necessary for the function of many, if not all GPCRs. Negative staining of detergent (n-dodecyl-b-D-maltoside)- solubilized disk membranes shows bi-lobed, roughly conical

˚

structures with lengths of 65 A, and these lobes are sepa-

˚

rated from each other by 32 A (Figure 1(g)).

Structure of Rhodopsin

Rhodopsin is a transmembrane protein consisting of an apoprotein, the 348 amino acid residue-long opsin

(Figure 2), linked to a chromophore, 11-cis-retinal. The chromophore is bound covalently via a protonated Schiff base to the Lys296-containing side chain of the opsin.

Bovine rhodopsin also is post-translationally modified. The N-terminal Met is acetylated, and Cys322 and

Cys323 of the C-terminus are palmitoylated, which

is very common in GPCRs. In addition, a disulfide bond exists between Cys110 (H-III) and Cys187 (E-II). Rhodopsin

is glycosylated at Asn2 and Asn15 by the hexasaccharide sequence (Man)3Glc(Nac)3. The molecular mass of bovine opsin with its post-translational changes (palmitoylation, acetylation of the N terminus and glycosylation) is 42 002.

Rhodopsin was crystallized from detergent solutions. The three-dimensional structure of bovine rhodopsin at

˚

2.8 A, the first high-resolution structure reported for a GPCR, reveals the internal organization of this receptor molecule (Figure 3). Rhodopsin folds into seven transmembrane helices (H-I–H-VII) that vary in length from 20 to 33 amino acid residues, and one cytoplasmic helix (H-8). The transmembrane residues are irregular and tilted at various angles due to their Gly and Pro residues. Further advances in rhodopsin crystallization were recently reviewed.

The N-terminal region of rhodopsin is located intradiscally (extracellular) and the C-terminal region is cytoplasmic (intracellular), each region possessing three interhelical loops. The extracellular region consists of four distorted b-strands, three interhelical loops and the doubly glycosylated N-terminal domain. The extracellular region from residues 173–198 acts as a plug for the chromophore-binding pocket (Figures 4(a) and 4(b)). The cytoplasmic surface contains 14 positively charged

residues, whereas the extracellular side contains only three positively charged residues, an arrangement that agrees with the positive inside rule for multispanning eukaryotic membrane proteins. The intracellular/cytoplasmic region of rhodopsin is essential for its vectorial transport from the site of synthesis to the ROS and it also plays important roles in G protein activation and photoactivated rhodopsin desensitization.

A highly conserved (D/E)R(Y/W) motif in the GPCR A family is formed by the tripeptide, Glu134-Arg135-Tyr136,

located in the cytoplasmic region of bovine rhodopsin (Figure 4(a)). The carboxylate group of Glu134 forms a

salt bridge with Arg135, a highly conserved residue among GPCRs, and Arg135 also interacts with Glu247 and Thr251 in H-VI. The ionization state of Glu134 is sensitive to its

environment, such that protonation of this residue causes rhodopsin activation (from meta IIa to meta IIb). This motif plays an important role in conformational changes in the structure of GPCRs that lead to their activation.

Another highly conserved NPXXY (Asn-Pro-Xaa-Xaa- Tyr) motif located at the end of helix VII and the beginning of H-8, is also close to the cytoplasmic region. Both the D (E)RY and NPXXY regions control the meta II (active) state of rhodopsin, and the NPXXY sequence is likely to be involved in G protein coupling (Figure 4(a)). The greatest distortion in H-VI is imposed by Pro267 (a highly

conserved residue among GPCRs) and H-VII is elongated due the presence of Pro291 and Pro303 (parts of NPXXY)

located in close proximity to Lys296, the retinal-binding residue. Highly conserved Glu122 and His211 are located

at the Zn2+-binding site. The interaction between the b ionone ring and H-III occurs at Glu122, one of the residues

that determine the rate of meta II decay (Figure 4(b)).

Chromophore-Binding Site

Visual pigments of both rod and cone cells contain the chromophore, 11-cis-retinal, bound covalently to a Lys side chain (Lys296 in bovine rhodopsin) via a protonated Schiff base. The absorption maximum (lmax) of free solubilized 11-cis-retinal is about 380 nm. When this chromophore binds to opsins, its lmax shifts toward longer wave lengths (a red shift) ranging from 435 nm (frog rods) to 560 nm (human cones). The protonated Schiff base linkage is responsible for about 70 nm of this shift. A further red shift results from the retinal-binding-pocket environment, especially its counter ion, which is Glu113 in vertebrate rhodopsins. In bovine rhodopsin, the lmax of mutant E113Q (Glu to Gln) is dramatically shifted from 498 nm to 380 nm. The absorption maximum also varies according to the interaction sites of the opsin molecule with the chromophore, especially dipolar interactions near the b ionone ring. Therefore, the lmax absorption of visual pigments varies from species to species that differ with respect to their opsin protein sequences.

The chromophore in rhodopsin is located in the core of the seven transmembrane helices, closer to the extracellular side of the disk membrane. 11-cis-Retinal helps maintain rhodopsin in an inactive state (Figure 4(b)). The retinalbinding pocket is formed by helices H-III, H-V, H-VI, and H-VII and the antiparallel b sheet of the N-terminal plug, part of extracellular loop II (E-II in Figure 2). Although the

retinal-binding site is very hydrophobic (Figure 4(b)), four charged residues, specifically Glu113 (H-III), Glu122 (H-III), Glu181 (b sheet), and Lys 296 (H-VII), are located near the

chromophore. Lys296 (H-VII) donates an amino group to form a protonated Schiff base and Glu113 (H-III), which is

˚

3.6 A away from the Schiff base, acts as a counter ion for this linkage. The positive charge on the protonated Schiff

base is energetically unstable in the hydrophobic protein core but the Glu113 (H-III) counter ion stabilizes the base by

shifting its pKa from neutral to alkaline. Highly conserved among all known vertebrate visual pigments, Glu113 (H-III) plays three important roles: (1) It keeps rhodopsin in its resting state by participating in the salt bridge with the Schiff base. Disruption of this bridge allows the H-VI motion that occurs upon photoactivation. (2) It prevents spontaneous hydrolysis of the Schiff base by stabilizing the protonated Schiff base via increasing its Ka by as much as 107. (3) It causes a major bathochromic (longer wavelength) shift in the maximum wavelength absorption of visual pigments. Longer wavelength absorption is essential because the front of the eye in most animals does not allow ultraviolet (UV) light to reach the retina. Steric hindrance, resulting either from mutating Gly121 (H-III) or substituting larger R groups at the C9 position, causes transducin (Gt) activation in the dark whereas lack of the C9 methyl group impedes photoactivation.

Opsin reacts within minutes with 11-cis-retinal to form rhodopsin. Similarly, 7-cis and 9-cis retinal also form visual pigments. In contrast, all-trans-retinal and 13-cis- retinal cannot regenerate opsin. The crystal structure of rhodopsin reveals that the chromophore-binding pocket is well defined, suggesting that the binding pocket has high specificity for the Schiff base and the b ionone ring. The exact location of these two components restricts the length of the chromophore-binding site. Therefore, 7-cis, 9-cis, and double and triple cis retinal analogs that have similar lengths and structures can regenerate the opsin, whereas chromophore analogs that are either shorter or longer than 11-cis-retinal cannot.

Rhodopsin Cycle – Retinal Isomerization

Inactive rhodopsin is activated upon light absorption, which induces a cis–trans isomerization that converts 11-cis-retinal to all-trans-retinal. The activation process can be divided into three phases (Figure 5).

1.Light-induced cis–trans isomerization of the retinylidene.

2.Thermal relaxation of the retinylidene–protein complex.

3.Hydrolysis of the Schiff base linkage, leading to formation of rhodopsin’s active meta II state.

Light absorption induces isomerization of 11-cis- retinylidene to all-trans-retinylidene, resulting in a transient intermediate called photorhodopsin, formed by the fastest chemical reaction (200 fs) in the rhodopsin cycle. Photorhodopsin is converted first into a thermally stable and high-energy intermediate product called bathorhodopsin. About 60% of the incident photon energy is stored in bathorhodopsin and then used to drive further conformational changes. In this state, the chromophore is in an 11-trans-15-anti conformation, a distorted all-trans conformation that results from steric restriction caused by the polyene chain of the retinal and the protein side chains. The b ionone ring and Schiff base are located

in a conformation similar to that of rhodopsin in the dark, but Thr181 and Glu113 are slightly moved. Then

the blue-shifted intermediate (BSI) is produced during the thermal relaxation of bathorhodopsin, but BSI can be observed only by time-resolved measurements during subsequent formation of lumirhodopsin. The distorted all-trans-retinal in bathorhodopsin relaxes by dislocation of the b ionone ring in lumirhodopsin. Displacement of this ring reflects the movement of helix III aided by interactions between other helices, that result in a slightly disordered structure during the transition from the dark state to lumirhodopsin. In particular, Thr181 and Glu122 (which are moved slightly in bathorhodopsin) become significantly moved due to the b ionone ring

displacement. Lumirhodopsin then relaxes further into meta I. Although the conformation of the chromophore in meta I closely resembles that in lumirhodopsin and the Schiff base proton is still hydrogen bonded, the overall structure is similar to rhodopsin. Meta I is further converted to meta II in two steps that can be separated by 20 ms in detergent-solubilized samples: (1) Conversion of meta I to meta IIa, accompanied by proton transfer from the Schiff base to the counterion Glu113. (2) Subsequent

uptake of a proton from the cytoplasm leading to meta IIb formation. The proton acceptor here is Glu134 (H-III), thus

meta IIa and meta IIb are in a pH-dependent equilibrium regulated by proton uptake at Glu134 (part of E/DRY motif). However, only meta IIb can trigger Gt activation. Deprotonation of the Schiff base is characterized by a large UV shift of the absorption maximum from 478 nm in meta I to 380 nm in meta II. A low-resolution crystal structure of a bovine-deprotonated Schiff base meta IIlike rhodopsin has been elucidated. Finally, meta II decays

into opsin and all-trans-retinal. Free opsin exhibits the largest conformational changes as compared to darkstate rhodopsin (Figure 5).

Visual Cycle – Rhodopsin Regeneration

The visual cycle consists of a series of reactions by which all-trans-retinal released from opsin isomerizes back into 11-cis-retinal that again binds to the opsin (Figure 5). This cyclic process does not require light. The released all-trans-retinal is transformed first to all-trans-retinol by a retinal dehydrogenase (RDH) in the ROS. The all-trans-retinol is transferred to the retinal pigment epithelium (RPE) where it is esterified by lecithin:retinol acyltransferase (LRAT), and later isomerized to 11-cis-retinol by a retinol isomerase. 11-cis-RDH then converts 11-cis- retinol back to 11-cis-retinal, which leaves the RPE to regenerate the opsin in the ROS.

Vertebrate versus Invertebrate

Rhodopsins

Enzymatic regeneration of rhodopsin does not occur in invertebrate visual systems where rhodopsin and metarhodopsin are photoconvertible. Upon photon absorption, 11-cis-retinal (or its analogs) of rhodopsin is converted into all-trans-retinal of metarhodopsin, and then irradiation of metarhodopsin changes the all-trans-retinal back to 11-cis-retinal by a process called photoregeneration.

Notably, invertebrates differ from vertebrates in the photoactivation of rhodopsin. Absorption of a photon by invertebrate rhodopsin leads to a stable meta II, but

retinal remains in the retinal-binding pocket. This contrasts to vertebrate rhodopsin where retinal leaves the meta II-binding pocket. Invertebrate phototransduction also involves an inositol-1,4,5-triphosphate signaling cascade (cyclic-guanosine monophosphate (GMP) in vertebrates) and a Gq-type G protein (Gt in vertebrates) that is stimulated by photoactivated rhodopsin.

Three-dimensional structures of bovine (vertebrate) rhodopsin and squid (invertebrate) rhodopsin show structural similarities in the arrangement of their transmembrane helices (Figure 6). Squid rhodopsin is a 50-kDa protein composed of 488 amino acid residues. A proline-rich 10-kDa C-terminal extension compared