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

sequence in the third helix which is involved in G protein interactions.

The presence of a proline-rich carboxyl-terminal tail is unique to cephalopod rhodopsins. This motif consists of 9–10 repeats of the pentapeptide Pro–Pro–Gln–Gly–Tyr that may facilitate receptor trafficking and morphogenesis. Though unique among rhodopsins, tandem repeats of proline-rich sequences are found in other protein families, where they are often associated with protein–protein interactions. Rhodopsin–rhodopsin interactions may be of structural importance in the cephalopod rhabdomere, since rhodopsin networks form in the microvilli of the rhabdomeral lobe. In native membranes, electron microscopy has revealed both poorly ordered rhodopsin clusters of 4–10 molecules and ordered rhodopsin pentameres. Intermolecular interaction is mediated in part by the rhodopsin carboxyl-termini, which aggregate and extend intracellularly from the membrane surface. In contrast, when the carboxyl-terminal is cleaved, crystalline lattice formation is observed both in reconstituted membranes and in crystallized rhodopsin. These more highly ordered crystalline arrays may be favored by interactions between transmembrane domains of adjacent molecules. It has therefore been suggested that the proline-rich region may function, in part, to limit crystalline array formation in native membranes, contributing instead to the formation of less-ordered rhodopsin clusters that confer membrane superstructure. Unfortunately, technical considerations prevent the observation of the squid rhodopsin crystal structure with an intact proline-rich tail, precluding definitive structural analysis.

The majority of opsins are covalently linked to an 11-cis-retinal chromophore and squid rhodopsin employs the 4-hydroxy-retinal derivative. In the squid, retinal is attached via a protonated Schiff ’s base linkage to a lysine residue in transmembrane domain 7 (residue 303 in Loligo and 305 in Todarodes). In contrast to the mammalian opsins, however, where the Schiff ’s base is stabilized using a glutamic acid residue of transmembrane domain 3 as a counterion, the crystal structure of Todarodes reveals the counterion to be a glutamic acid at residue 180, which is located between transmembrane helices 3 and 4.

On absorption of a photon, 11-cis-retinal isomerizes to all-trans-retinal in a few hundred femtoseconds, which induces conformational changes in the opsin component. In invertebrates, the product of photoexcitation is an active metarhodopsin that is comparable in stability to inactivated rhodopsin. This contrasts the situation in vertebrates, where rhodopsin undergoes rapid sequential transitions between several unstable intermediate states. In vertebrates, all-trans-retinal is released during thermal relaxation of photoexcitation products and rhodopsin must be regenerated through subsequent recombination of opsin with another molecule of 11-cis-retinal. In the squid, however, the retinoid chromophore can remain attached to opsin throughout the rhodopsin activation cycle, and in a subsequent deactivating photoconversion event, invertebrate metarhodopsin can be converted to inactive rhodopsin by photon absorption. Thus, photobleaching may not be inevitable in the squid retina. The spectral sensitivities of squid rhodopsin and metarhodopsin differ by only a few nanometers (493 and 500 nm,

respectively) suggesting that the same visual stimulus could be both activating and inactivating. Due to the comparable stabilities and interconverting wavelengths of rhodopsin and metarhodopsin, it has been suggested that substantial populations of each could exist at steady state in the squid eye. However, retinas obtained from freshly caught squid are found to contain almost exclusively rhodopsin, a finding that suggests a highly efficient inactivation pathway.

In addition to rhodopsin, cephalopod photoreceptor cells contain retinochrome, a second photosensitive retinal-binding protein implicated in rhodopsin regeneration. Squid retinochrome from Todarodes pacificus has been cloned, revealing a 301-amino-acid protein. The structure of retinochrome resembles that of rhodopsin, but its retinoid occupancy is reversed; whereas rhodopsin binds 11-cis-retinal and produces all-trans-retinal on photoisomerization to metarhodopsin, retinochrome binds and photoisomerises all-trans-retinal to 11-cis-retinal in its conversion to metaretinochrome. Metaretinochrome then releases 11-cis-retinal, providing it to rhodopsin via a shuttling protein known as retinal-binding protein (RALBP). In the dark, metaretinochrome is localized in the arhabdomeral lobe, where it releases 11-cis-retinal and, subsequently, binds all-trans-retinal released from metarhodopsin. Soluble RALBP then shuttles 11-cis-reti- nal to the rhabdomeral microvilli, where it binds retinalfree opsin, which may have arisen as a photoproduct or by synthesis de novo, in order to generate rhodopsin. Lightdependent translocation of both rhodopsin and retinochrome has been documented. In the dark, rhodopsin and retinochrome colocalize at the base of the microvilli, while in the light rhodopsin redistributes along the entire area of the microvillar membrane, and retinochrome becomes more plentiful in the rhabdomeral lobe. Dynamic control of the availability of rhodopsin (and other lightabsorbing pigments and signaling proteins) in signaling compartments may modulate the cascade and rhodopsin regeneration. Thus, the eye can adjust to dim or bright ambient light conditions, accurately perceive objects in each, and regenerate rhodopsin when necessary.

Squid Visual Guanine Nucleotide-Binding

Protein, Gq

Squid rhodopsin couples to its effector enzyme, a PLC, via a heterotrimeric G protein belonging to the Gq subfamily. Like all G proteins in this family, squid Gq is composed of three nonidentical subunits, a, b, and g. These subunits are associated with each other in the inactive state with GDP bound to the a subunit. In this state, the G protein is tightly bound to the rhabdomeric membrane, likely in association with rhodopsin. Upon 11-cis-retinal isomerization by light, a conformational change in the receptor causes a

conformational change in the G protein subunits, which opens up the gunanine nucleotide-binding site on the a subunit allowing GDP to be exchanged for GTP. In the GTP-bound state, Gqa has a lower affinity for rhodopsin and its bg partners, but gains a higher affinity for its effector, PLC. PLC is recruited to the rhabdomeric membrane to interact with activated Gqa and its substrate phosphotidylinositol 4,5-bisphosphate. Gqa binding activates the PLC enzyme and at the same time the PLC stimulates the GTPase activity of Gqa resulting in hydrolysis of the terminal phosphate group on the bound GTP, thus rendering the G protein once again inactive in the GDP-bound state. Reassociation of the G protein subunits completes the cycle of activation and inactivation back to the basal state. The G protein is then ready to receive the next signal from rhodopsin.

Gq protein subunits have been purified and cDNA sequences encoding the proteins have been reported from L. forbesi and L. pealei. Loligo Gqa is similar in amino acid sequence to Gqa proteins of other species with a conserved guanine nucleotide-binding domain and three switch regions that are the major sites of conformational change in the protein when GDP is exchanged for GTP on the protein. The sites of interaction between a G protein a subunit and its receptor are primarily in the two ends of the protein. The carboxyl-terminus of squid Gqa is identical to that found in Gq proteins from other species; however, the amino-terminus of the protein in all invertebrates lacks a six-amino-acid extension found in mammalian Gqa. Studies using L. pealei Gqa expressed in mammalian cells suggest that the modified amino-terminus found in invertebrate proteins increases the efficacy of G protein activation by receptors.

The amino-terminus is also the site of posttranslational addition of palmitic acid on one or more of the two cysteine residues at position 3 and 4 of the Gqa subunit. This lipid modification helps maintain membrane association of the Gq protein and is particularly important for keeping Gqa attached to the membrane following activation by the receptor when Gqa dissociates from the receptor and Gqbg subunits.

The Gqbg subunits have not been examined extensively. They appear to have similar functions to other G protein bg subunits in that they associate with the Gqa subunit when it is bound to GDP and dissociate when Gqa is bound to GTP. The bg subunits are tightly associated with the retinal membranes at all times and lipid modifications of the g subunit may contribute to this localization. Loligo Gb has a similar sequence to that of all other G protein b subunits, whereas Loligo Gg is quite distinct from Gg subunits of other species. Gqbg subunits do not activate purified PLC from squid eyes. Further studies will be required to determine if squid G protein bg subunits have any additional roles in visual signal transduction.

Squid Visual PLC

The protein stimulated by activated Gq in the squid visual system is PLC. The protein has been purified and the amino acid sequence determined from L. pealei. Immunoblot analysis of many squid tissues showed that the visual PLC is uniquely expressed in the photoreceptor membranes. Squid visual PLC is a 140 kDa protein that has significant sequence similarity and a domain structure that is common to phospholipase b enzymes; a PH domain that helps the protein bind to membranes, X and Y catalytic domains, and a C2 domain that is likely the site of calcium binding to the enzyme. In the absence of light stimulus to the photoreceptors, intracellular calcium concentrations are low and the PLC has very little catalytic activity. Upon activation of rhodopsin, the catalytic activity of the phospholipase is highly stimulated by the binding of activated Gqa to domains in the carboxyl-terminal end of the protein known as P and G boxes. The enzyme hydrolyzes membrane phospholipids with preference for phosphatidylinositol 4,5-bisphosphate, which is converted into inositol 1,4,5-trisphosphate (IP3) and DAG. A network of submicrovillar tubules has been observed beneath the microvilli that may express IP3 receptors and release stored calcium in response to IP3 activation. A rapid rise in intracellular calcium may help to maintain PLC activity as these enzymes are stimulated in the presence of elevated calcium concentrations.

In addition to the role that calcium plays in visual signal transduction, high (millimolar) calcium concentrations can activate a calpain-like protease found in the squid retina. This protease can cleave several proteins in the visual signaling pathway. Calpain cleaves PLC near the carboxyl-terminus and renders the protein insensitive to Gqa activation. The protease can also cleave rhodopsin near its carboxyl-terminus removing the proline-rich repeat sequence from the rest of the molecule. Calcium activation of this protease may therefore play a role in freeing rhodopsin and Gq to allow for greater membrane turnover following prolonged light exposure.

Light-Activated Ion Channel

The final component in the visual signal transduction system is the channel regulated by PLC hydrolysis of membrane lipids. This component of the squid visual system has been characterized in only one report; an electrophysiological recording made from an isolated L. pealei photoreceptor. Light stimulation of the squid photoreceptor evoked an inward current with a peak amplitude greater than 1000 pA and channel activity was most frequently recorded when the patch electrode was placed near the apical tip of the cell where microvilli are present.

A squid photoreceptor channel has been identified and cloned from L. forbesi and it was found to be most homologous to that of the visual transient receptor potential (TRP) ion channels identified in Drosophila. TRP and an additional TRP-like channel have been shown to constitute the light-activated response in Drosophila. Analysis of the amino acid sequence of the squid channel suggests a protein with six or eight membrane spanning segments. The amino-terminus contains an ankyrin-like repeat sequence similar to that found in Drosophila TRP channels, and may account for the association of the protein with the cytoskeleton during purification. The carboxyl-terminus of the squid channel is considerably shorter than that found in the Drosophila TRP channel and lacks the pro- line-rich repeat sequence suggested to link TRP to intracellular Ca2+ stores. Expression of a peptide composed of the squid channel carboxyl-terminus bound to calciumcalmodulin in vitro. These studies suggest that the squid channel may be regulated differently from that of Drosophila, however, since the squid channel has not yet been characterized, its properties and the mechanisms by which it is regulated by the PLC signaling cascade remain speculative.

Desensitization of Visual Signal

Transduction

Once rhodopsin has been activated, a series of protein–protein interactions occur within the photoreceptor cell to terminate signal transduction and restore the photoreceptor to its inactive state. Like most GPCRs, activated squid rhodopsin is phosphorylated by a G-protein- coupled receptor kinase (GRK) also called rhodopsin kinase. Light-activated rhodopsin also binds arrestin and biochemical studies using purified arrestin have demonstrated that this uncouples rhodopsin from activation of Gq.

Squid Rhodopsin Kinase

The squid visual system expresses a kinase that has sequence and functional similarity with other GRKs. The most extensively studied GRKs are the mammalian rhodopsin kinases (GRK1 and GRK7) and b-adrenergic receptor kinases (GRK2 and GRK3). Interestingly, molecular cloning of squid rhodopsin kinase (SQRK) revealed much higher sequence similarity to the mammalian b-adrenergic receptor kinase GRK2 (66%) than to GRK1 (33%), which terminates signaling in the mammalian visual system. This is a common theme among invertebrate rhodopsin kinases, as eye-specific GRK1 cloned from Drosophila and octopus rhodopsin kinase (ORK) also bear higher sequence identity to GRK2 than to GRK1. The structural similarities between SQRK and GRK2 include a central

serine/threonine kinase catalytic domain, a structurally conserved amino-terminal domain bearing an RGS domain, a conserved carboxyl-terminal sequence and a PH domain in the carboxyl-terminal. In GRK2, it has been established that a carboxyl-terminal region partially overlapping the PH domain associates with the G protein bg subunits hence facilitating membrane localization in a stimulus-dependent manner. These structural similarities suggest that the phosphorylation of squid rhodopsin may more closely resemble the phosphorylation event of the mammalian b-adrenergic receptor than that of mammalian rhodopsin. SQRK structural motifs bearing high homology to the GRK2 Ca2+/CaM-binding domain and the GRK2 clathrin box motif suggest that Ca2+ may have a role in regulating SQRK activity and that SQRK may also bind to the clathrin heavy chain and play a role in endocytosis. To date, functional characterization of SQRK has confirmed that purified SQRK is able to phosphorylate squid rhodopsin in rhabdomeric membranes in a light-dependent manner. SQRK phosphorylation of rhodopsin requires GTP and Mg2+ ion cofactors that may relate to the need to activate the Gq protein to allow SQRK access to rhodopsin.

Squid Visual Arrestin

Squid visual arrestin (sArr) has been cloned, purified, and characterized with respect to its functional interactions with rhabdomeric membranes. Squid arrestin from L. pealei is a 400-amino-acid protein with an estimated mass of 55 kDa that is expressed exclusively in eye tissue. Sequence identity between sArr and those from Drosophila and Limulus are 42% and 37%, respectively (sequence similarity including conservative substitutions is considerably higher, at 61% and 60%, respectively). Of the mammalian arrestins, visual arrestin from L. pealei shares highest identity with b-arrestins (44% identity and 64% similarity to b-arrestin1; 42% identity and 63% similarity to b-arrestin2). Squid visual arrestin is only 32% identical to mammalian visual arrestin, conservatively substituted to 49% similarity. This pattern of similarity parallels that between invertebrate rhodopsin kinase and mammalian b-adrenergic receptor kinases, and suggests that the functional interactions with invertebrate rhodopsin resemble those of the mammalian b-adrenergic receptor more closely than those of mammalian rhodopsin.

For many GPCRs, including mammalian rhodopsin, it has been demonstrated that receptor phosphorylation enhances high-affinity binding of arrestin. Accordingly, the primary structure of squid arrestin contains both conserved residues associated with both highand lowaffinity phosphate interactions. However, purified sArr does not seem to require rhodopsin phosphorylation to

bind light-activated rhodopsin. This parallels biochemical studies in Drosophila, where purified Arr2 can bind to phosphorylated and unphosphorylated light-activated rhodopsin with comparable affinity. Further, light-dependent binding of Arr2 was equivocal in wild type and mutants where rhodopsin cannot be phosphorylated (both a truncation mutation lacking the phosphorylation site, and a serine to alanine point mutation that retains a similar structure but which cannot be phosphorylated). These findings are consistent with observations that the phosphorylation-deficient rhodopsin mutants display similar deactivation kinetics. Thus for invertebrates, the role of rhodopsin phosphorylation in arrestin binding is unclear, and light activation of rhodopsin may be sufficient.

Squid visual arrestin associates with the rhabdomeric membrane in a light-dependent manner and inhibits light-activated GTPase activity. This is consistent with the notion that recruitment and stoichiometric binding to the intracellular surface can uncouple Gq from the receptor and terminate signaling by a competitive mechanism. In the dark, squid arrestin also has appreciable affinity for the rhabdomeric membrane, an interaction which can be abrogated by inositol 1,2,3,4,5,6-hexakisphosphate (IP6), a soluble analog of the membrane lipid phosphatidylinositol 3,4,5-triphosphate. This suggests that arrestin can also bind to membrane phospholipids, an interaction that studies in Drosophila suggest may mediate arrestin trafficking along an elaborate system of cytoplasmic structural components.

The primary structure of sArr includes five fingerprint motifs that correspond with domains of distinct functional importance conserved among the arrestin family; a region that recognizes receptor activation, a domain rich in hydrophobic interactions, a domain that recognizes receptor phosphorylation, a carboxyl-terminal regulatory domain, and a conserved amino-terminal domain. Overall, the arrestin molecule adopts a concave saddle-like conformation consisting of aminoand carboxyl-terminal domains rich in antiparallel b-sheets that hinge on a central polar core region of buried salt bridges that are disrupted when the molecule encounters the activated receptor. The primary sequence of sArr contains 26Asp, 169Arg, 293Asn, 300Asp, and 381Arg, which are identical to bovine visual arrestin (except 293Asn, which is a conservative substitution). These five essential conserved residues are contained within consensus sequences of five to eight identical or conservatively substituted amino acids that form the salt bridges buried in the polar core. Both the aminoand carboxyl-terminal domains mediate high-affinity binding to rhodopsin intracellular loops and these regions are located exclusively on the concave side of arrestin. Arrestin binding to rhodopsin thus occludes the binding of the G protein, preventing catalysis of GTP exchange, and uncoupling the receptor from its effector.

Interestingly, sArr can be phosphorylated by SQRK, a novel function among the GRK family. This contrasts the phosphorylation of Drosophila and Limulus visual arrestins, which are phosphorylated by Ca2+/calmodulin-dependent kinase II. Phosphorylation of purified sArr requires SQRK, membranes, light-activated rhodopsin, and the presence of Ca2+. Though the kinase is different, Ca2+ dependence is common to Limulus and Drosophila. Studies in Drosophila show that the kinetics of invertebrate visual arrestin phosphorylation are fast (seconds after illumination, 43% of Drosophila Arr2 is phosphorylated) and that arrestin phosphorylation can facilitate its release from rhodopsin once arrestin-bound metarhodopsin is photoconverted back to its inactive state. A similar role for squid arrestin phosphorylation has been found, as phosphorylated arrestin dissociates from dark-adapted membranes more readily than unphosphorylated arrestin.

Conclusion

Many of the molecular components of the squid visual system have been identified and characterized. Studies have suggested many similarities between the squid and other invertebrate visual systems. The strength of the squid system has been in the abundance of retinal tissue that makes protein purification and characterization feasible. These biochemical studies have complemented the power of genetic manipulations in the Drosophila system and the ease of electrophysiology in Limulus. Further studies are still required to determine the roles of rhodopsin and arrestin phosphorylations as well as identification of the many proteins involved in resetting the signaling components in the dark. We still know very little about how the activation of PLC results in membrane depolarization and the characteristics of the ion channels in the microvillar membranes. Future studies may eventually reveal all the molecular machinery of this fascinating visual system.

See also: Circadian Photoreception; Genetic Dissection of Invertebrate Phototransduction; Microvillar and Ciliary Photoreceptors in Molluskan Eyes; Phototransduction in Limulus Photoreceptors; Phototransduction: Inactivation in Cones; Phototransduction: Inactivation in Rods; Phototransduction: Phototransduction in Cones; Phototransduction: Phototransduction in Rods; Phototransduction: Rhodopsin; Evolution of Opsins; The Photoreceptor Outer Segment as a Sensory Cilium.

Further Reading

Go, L. and Mitchell, J. (2007). Receptor coupling properties of the invertebrate visual guanine nucleotide-binding protein iGqa. Cellular Signalling 19: 1919–1927.

Mayeenuddin, L. H., Bamsey, C., and Mitchell, J. (2001). Retinal phospholipase C from squid is a regulator of Gq alpha GTPase activity. Journal of Neurochemistry 78: 1350–1358.

Mayeenuddin, L. H. and Mitchell, J. (2001). cDNA cloning and characterization of a novel squid rhodopsin kinase encoding multiple modular domains. Visual Neuroscience 18: 907–915.

Monk, P. D., Carne, A., Liu, S.-H., et al. (1996). Isolation, cloning and characterization of a trp homologue from squid (Loligo forbesi) photoreceptor membranes. Journal of Neurochemistry

67: 2227–2235.

Murakami, M. and Kouyama, T. (2008). Crustal structure of squid rhodopsin. Nature 453: 363–367.

Nasi, E. and Gomez, M. (1992). Electrophysiological recordings in solitary photoreceptors from the retina of squid, Loligo pealei. Visual Neuroscience 8: 349–358.

Ryba, N. J., Findlay, J. B., and Reid, J. D. (1993). The molecular cloning of the squid (Loligo forbesi) visual Gq-alpha subunit and its expression in Saccharomyces cerevisiae. Biochemical Journal

292: 333–341.

Swardfager, W. and Mitchell, J. (2007). Purification of visual arrestin from squid photoreceptors and characterization of arrestin interaction with rhodopsin and rhodopsin kinase. Journal of Neurochemistry 101: 223–231.

Venien-Bryan, C., Davies, A., Langmack, K., et al. (1995). Effect of C-terminal proline repeats in ordered packing of squid rhodopsin and its mobility in membranes. FEBS Letters 359: 45–49.

Walrond, J. P. and Szuts, E. Z. (1992). Submicrovillar tubules in distal segments of squid photoreceptors detected by rapid freezing.

Journal of Neuroscience 12: 1490–1501.

Glossary

Avoidance of saturation – The ability of cones (but not rods) to continue functioning in steady background illumination of arbitrarily high intensity. Light adaptation – The rapid adjustment of sensitivity and kinetics (of the entire visual system, or of the photoreceptors) that occurs in response to altered ambient light intensity. The adjustment is rapid irrespective of whether the change is an increase or decrease in intensity, provided that the change is not too great.

Saturation – The failure of the photoreceptors (or of the visual system) to respond to incremental illumination in the presence of appropriately bright illumination. The rod photoreceptors saturate at a relatively low background intensity; in contrast, the cone photoreceptors avoid saturation by steady backgrounds, no matter how bright the light is. Weber’s law – The reduction in visual sensitivity that occurs in inverse proportion to the intensity of the ambient background illumination. This corresponds to a rise in visual threshold in direct proportion to the ambient illumination.

Performance of the Photopic (Cone)

System

Workhorse of Vision

For human vision, the photopic cone system can be considered the workhorse of vision, because it is operational under almost all of the conditions that we (in a modern world) experience. Thus, it is our photopic system that provides our sense of vision under all lighting conditions, apart from exceptionally low levels such as starlight conditions. Under moonlight conditions, our scotopic and photopic systems are both functional, over an intensity range that is termed mesopic. If you are ever in doubt as to whether you are using your photopic system under twilight or nighttime conditions, there is a simple test: if you are able to detect any color in the scene, then your cones are active; your rods may also be active, but this is one test of whether there is any cone activity at that level of intensities. In addition, the photopic system remains functional at all higher intensities, up to the brightest sunlit conditions than we ever experience.

It is interesting to consider that, despite their enormous importance to our vision, cones make up perhaps only 5% of the population of photoreceptors over most of our peripheral retina. This relatively low proportion of cone photoreceptors in the peripheral retina is entirely adequate for our normal peripheral vision, which requires only relatively low spatial acuity. Even though the great majority of peripheral photoreceptors are rods, they are simply not used under most of the circumstances that we think of as vision – thus, they are only used at exceedingly low ambient lighting levels. The reason for the great numerical preponderance of rods is to be able to capture every available photon at those very low light intensities.

Rapid Response and Moderate Sensitivity

In survival terms, one of the greatest advantages of cones over rods is their much faster speed of response. The responses of our rods, even when they are light adapted, are much too slow to allow us to function visually at the speeds that are required to escape predators and to capture prey. Cones, instead, are specialized so as to permit extremely rapid signaling of visual stimuli to the brain.

Cones are often described as having much lower sensitivity than rods, but this view is misleading, especially when considered in terms of the rapidly changing visual stimuli that the cones are specialized for signaling. Although the peak sensitivity to a brief flash of light may be perhaps 30-fold lower in a cone than in a rod, the sensitivity to rapidly fluctuating stimuli is considerably higher in cones than in rods; thus, the slow response of the rods makes them quite insensitive to rapidly changing stimuli. When expressed in terms of the efficacy of activation within the G-protein cascade of phototransduction, the amplification in cones and rods appears to be essentially indistinguishable – the real difference is in the speed of inactivation.

Avoidance of Saturation

The second crucial feature of cones, in terms of survival advantage, is their amazing ability to avoid saturation no matter how intense the steady background illumination becomes. This property stands in stark contrast to the situation in rods, which are completely incapable of responding once the background exceeds a relatively low level (corresponding roughly to twilight illumination). One of the major challenges in photoreceptor research is

to provide a clear understanding of how it is that cones are able to avoid saturation at arbitrarily high light intensities, whereas rods succumb to saturation at very low light intensities. As will be described below, considerable advances have recently been made toward providing this understanding.

Light Adaptation of the Cones

Cone photoreceptors undergo light adaptation over an enormously wide range of intensities, and it is likely that almost all of the adaptation that is observed in the overall photopic visual system is mediated by these changes occurring at the level of the receptor cells. This section describes the adaptational effects that occur in the cone photoreceptors.

Flashes on Backgrounds: Desensitization and Acceleration

Figure 1 illustrates the responses of a cone photoreceptor to the same set of flashes presented under three different conditions; the flashes A–F were of progressively greater intensity from left to right, but were exactly the same in each of the three panels. In (a), the flashes were presented in darkness, and represent a standard dark-adapted flash family; in (b), the same flashes were presented shortly after a dim steady background had been turned on; and in (c), the same flashes were presented after the onset of a brighter background. In the presence of the background illumination, the amplitude of the responses to dim flashes was smaller. For example, for the second flash intensity, B, the response amplitude becomes markedly smaller from

(a) to (b) to (c). In other words, backgrounds of increasing intensity progressively desensitized the cone’s incremental

8.8 103

Light

8

Sensitivity units)(arbitrary 4 0

Time (s)

Figure 2 Incremental responses of a salamander cone to test flashes presented on backgrounds of increasing intensity. The largest trace is for a dim flash presented in darkness, while the other traces correspond to the same test flash presented on backgrounds. In fact, in the presence of brighter backgrounds, the test flash intensity was increased in order to obtain measurable responses, and the plotted traces have therefore been scaled as response divided by test flash intensity, so as to provide a direct measure of sensitivity. Reproduced with permission from Matthews, H. R., Fain, G. L., Murphy, R. L. W., and Lamb, T. D. (1990). Light adaptation in cones of the salamander: A role for cytoplasmic calcium concentration.

Journal of Physiology 420: 447–469.

response. Such behavior is very characteristic of photoreceptors, and these responses from cones are qualitatively similar to those obtained from rods.

The manner in which the response to a dim flash is modified by the presence of backgrounds of different intensity is illustrated in Figure 2. The largest trace is the response to a dim flash presented under fully darkadapted conditions, while the other traces are for the same flash presented on backgrounds of progressively brighter intensity. (In fact, in order to maintain responses of measurable amplitude, the flash intensity was increased in the presence of backgrounds, and the traces actually plot response divided by flash intensity; i.e., the response sensitivity.)

The traces in Figure 2 demonstrate that the effect of backgrounds of increasing intensity is to both desensitize and accelerate the response to an incremental dim flash. This behavior of cones is very similar to that exhibited by rods.

Dependence of Sensitivity on Background Intensity: Weber’s Law

By plotting the peak amplitude of each of the traces in Figure 2 as a function of the background intensity on which it was measured, one obtains a sensitivity versus background plot of the type illustrated in Figure 3.

The results plotted in Figure 3 were obtained over an extremely wide range of background intensities by Dwight Burkhardt using a laser source of illumination. Importantly, the preparation was the intact eyecup (of the

−8 −9

−10−2 −1 0 1 2 3 4 5 6 7 8 9 10 11 Log background intensity (photons s−1 μm−2)

Figure 3 Cone sensitivity as a function of background intensity. Data are plotted in double logarithmic coordinates, and were obtained from intracellular measurements averaged from 15 cones in the turtle eyecup preparation. The experiments used laser illumination to achieve very high background intensities, and monitored step sensitivity rather than the more conventional flash sensitivity. The smooth curve plots Weber’s law, given by eqn [1]. Reproduced from Burkhardt, D. A. (1994). Light adaptation and photopigment bleaching in cone photoreceptors in situ in the retina of the turtle. Journal of Neuroscience 14: 1091–1105.

turtle), so that the photoreceptors remained in contact with the retinal pigment epithelium (RPE) and thereby experienced normal regeneration of visual pigment, in order that meaningful results could be obtained even at very high background intensities. (One methodological difference between the results plotted in Figure 3 and the results that are more usually plotted is that step sensitivities rather than flash sensitivities are plotted; however, this does not, in practice, make much of a difference.)

For background intensities from 103 to 1011 photons mm 2 s 1, the relationship between log sensitivity and log background intensity is a straight line with a slope of 1; in other words, over roughly 8 log units of background, the turtle cone’s sensitivity declines inversely with background intensity. The curve plotted near the points in Figure 3 represents Weber’s law, described by:

where S is flash sensitivity, SD is its dark-adapted value, I is the background intensity, and I0 is the half-desensitizing intensity, also known as the dark-adapted equivalent background intensity. The good fit of the Weber’s law expression shows, very importantly, that cone photoreceptors in the intact eyecup are able to completely avoid saturation, even at enormously high intensities of steady illumination. This feature represents a crucial distinction between the properties of cones and rods. The circulating current of rods is shut off at quite low background intensities, so that

the rods are unresponsive to superimposed stimuli, in the presence of background illumination of moderate intensity.

In the same set of experiments, Burkhardt measured the intensity at which 90% of the pigment was in the bleached state, and found this to be around 106 photons mm 2 s 1. For all intensities above that level, the observed Weber’s law behavior can be accounted for in terms of pigment bleaching. For each additional 10-fold increase in intensity, there will be a 10-fold reduction in the amount of pigment remaining available to absorb light, and hence there will necessarily be a 10-fold reduction in sensitivity in the absence of any other change of parameters of transduction in the outer segment. In other words, if the photoreceptor is able to avoid saturation up to intensities that cause substantial bleaches, then it will be able to exhibit Weber’s law desensitization at higher intensities purely by means of pigment bleaching. Cones are able to function up to this critical intensity, whereas rods saturate at much lower intensities.

Extremely Rapid Recovery of Cone

Photocurrent

In order to measure the performance of mammalian cones in the presence of extremely bright background illumination, it is necessary to use a preparation in which the cones

are in contact with the RPE (as was the case in the experiments above with turtle cones). Accordingly, it is not appropriate to use suction-pipette experiments at very high intensities. On the other hand, experiments measuring the electroretinogram (ERG) in the intact eye are very suitable.

Results from an experiment designed to measure the kinetics of recovery of the circulating current of human cone photoreceptors, upon extinction of steady illumination that bleached 90% of the visual pigment, are illustrated in Figure 4. This Figure shows recordings of the a-wave of the human ERG, which monitors primarily the response of photoreceptors; and at these incredibly high intensities, only the cone photoreceptors are responding. The left panel shows the response to a bright flash superimposed on the intense steady background, while the right panel shows the response obtained at extinction of that background. As indicated by the right-hand pair of vertical scales, the bright flash responses established the zero level of circulating current, as well as the level of circulating current during the intense background (i.e., unity on the inner scale). Separate measurements (not shown) established the dark current (i.e., unity on the outer right-hand scale). Even during the presence of the intense steady background, the cone circulating current was roughly 50% of its original level in darkness.

ERG (μV)

40

b

30

20

10 a

0

−10

−20

0 5 10 15

Time after flash (ms)

Time after background extinction (ms)

The traces on the right show the ERG a-wave upon extinction of the intense background. Little change occurs for the first 7 ms, but thereafter a substantial upward response occurs, the OFF a-wave indicated by the blue arrow, until about 15 ms after extinction of the background; at this point, the a-wave is obscured by spikelike activity of the b-wave. There is compelling evidence that the a-wave traces for these subjects monitor the recovery of the cone circulating current. On this basis, the cone circulating current is essentially fully recovered within about 15 ms after extinction of illumination so intense that it bleaches 90% of the cone pigment. This is extremely rapid recovery, at least when compared with the time course of recovery following intense flashes delivered from darkness. The next section considers the speed that is required for the shut-off reactions of phototransduction, in order to be able to account for recovery of the circulating current as fast as is shown in Figure 4. The smooth gray curve near the measured traces in the righthand panel of Figure 4 was calculated from the model presented in the next section, using the short time constants listed in Table 1.

Extremely rapid recovery of cone circulating current, as inferred from the results of Figure 4, is also required in order to account for classical experiments on the flickerfusion frequency of human subjects. Even at quite low photopic intensities, human subjects are able to detect square-wave flicker at a frequency of around 50 Hz using peripheral vision. However, at higher intensities, the flicker-fusion frequency increases to 100 Hz or more. At a frequency of 100 Hz, the illumination is being switched on and off at intervals of 5 ms each. Thus, in

order for the flicker to be detectable, some degree of recovery of cone circulating current must have occurred within 5 ms. Hence, this finding is broadly consistent with the time course inferred from Figure 4.

Molecular Basis of Cone Light Adaptation

Reaction Steps Underlying Rapid Recovery of

the Cone’s Light Response

Figure 5 presents a schematic of the reaction steps underlying phototransduction in cones, where shut-off reactions and the lifetimes (or turnover times) of important intermediates are indicated in red.

For mammalian cones, the speed of the various shut-off reactions shown in the schematic of Figure 5 has been estimated in a number of recent studies using intact preparations. In the case of monkey cones, the parameters were extracted through theoretical modeling of results obtained from intracellular recordings of horizontal cells in the retina–RPE–choroid preparation. In the case of human cones, the parameters were extracted through theoretical modeling of ERG results, including those of the type illustrated in Figure 4. The shut-off reactions have been found to be extremely rapid, and the parameters that have been reported are summarized in

Table 1.

The collected estimates in Table 1 are consistent with the notion that all four of the shut-off time constants in human cones are extremely short, with values in the range of 3–18 ms; in fact, it appears that three of the time constants could be around 5 ms or less, and one around