Proteins that stabilize the structure of outer-segment discs

Rhodopsin is not only integral to the phototransduction cascade, it is also required for the formation and maintenance of OS discs; in the absence of rhodopsin, the OS structure is not formed.²² Peripherin and Rom-1^15,34,35 are two other disc membrane components that contribute to maintaining the flattened structure of the disc. Both of these proteins belong to the tetraspanin family of integral membrane proteins that form large multiprotein complexes known as the tetraspanin web or tetraspanin-enriched microdomains.³⁶ Peripherin/rds is normally found within the edge or “rim” of the disc. It self-associates to form higher-order complexes and also interacts with Rom-1.³⁷ Peripherin/rds and Rom-1 likely function as adhesion molecules that assist in keeping the discs vertically aligned, and may stabilize the disc stack with bridges to the overlying plasma membrane. Peripherin/rds is also thought to function in forming the curvature of the disc rim.³⁸

Deletion or disruption of peripherin/rds results in malformation of discs at the OS base. The naturally occurring retinal degeneration in the mouse named “retinal degeneration, slow” (rds) has been shown to be due to a defect in peripherin/rds. The absence of peripherin/rds in the rds/rds mouse prevents normal development of the photoreceptor OS and leads to photoreceptor cell death.^15,39 Transgenic mice that lack peripherin/rds fail to form an OS. When the level is reduced, as in the heterozygous mice, large whorls of disc membrane are formed instead of an organized stack of uniform discs.^15,39 Mutations that affect the quaternary structure of peripherin/rds also lead to malformed discs. These observations underscore the importance of peripherin/rds in the formation and maintenance of disc structure in the OS.

Although Rom-1 is analogous to peripherin in structure and function, its absence in rods shows a less severe phenotype than peripherin/rds knockout mice, suggesting that peripherin/rds is more critical for disc formation. Although the discs grow larger than normal sizes, photoreceptor function appears to be retained to a much higher level than when peripherin/rds is absent. Nevertheless, abnormal Rom-1 does lead to slow and progressive photoreceptor degeneration both in mice and in humans.⁴⁰

Functional differences of peripherin/rds and Rom-1 have been noted in rods and cones. Cones appear to have a lower ratio of Rom-1/peripherin,³⁶ and different peripherin/rds mutations appear to affect rods and cones differentially. For example, the C214S and N244K mutations have a greater effect on rods whereas R172W and N244H tend to cause cone-dominant diseases such as macular dystrophy.³⁶

Transmission electron microscopy has revealed structural elements between the lamellar discs as well as between the discs and the plasma membrane that are mainly localized to the rim region and incisures of discs.^41–44 A study of OS structure using cryoelectron tomography of vitrified OS showed previously unobserved spacers distributed throughout the discs.³⁰ These structural elements likely maintain the proper distance between adjacent discs and between discs and the plasma membrane. The protein identities of these spacers are not known, but peripherin/rds and Rom-1 may be candidates.

Another component of the rod disc is ABCR (photoreceptor cell-specific ATP-binding cassette transporter).^45–47 Mutations in ABCR are responsible for a large variety of retinal degenerations, including Stargardt macular dystrophy, fundus flavimaculatus, some forms of cone–rod degeneration, and retinitis pigmentosa (RP). Other ABCR mutations are thought to increase the risk of developing age-related macular degeneration (AMD). ABCR is a member of the ABC superfamily. This is a large family of transmembrane proteins involved in energy-dependent transport of many different substrates across membrane “barriers.” The localization of ABCR at the disc rim suggests that it is involved in the movement of molecules from the disc lumen into the cytosol. This hypothesis was supported by studies in ABCR knockout mice, which show delayed dark adaptation, increased all-trans retinal following light exposure, and elevated phosphatidylethanolamine (PE) in the rod OS.⁴⁸ Biochemical analysis of retinas from ABCR knockout mice revealed accumulation of a novel complex of all-trans retinal and PE, termed N-retinylidene-PE, which is not found in normal retina. Once in the cytosol, the all-trans retinol moves to the retinal pigment epithelium (RPE) for recycling and chromophore (11-cis retinal) regeneration (see Chapter 16, Cell biology of the retinal pigment epithelium). The primary pathologic defect in Stargardt disease, and also in ABCR knockout mice, is accumulation of toxic lipofuscin pigments, such as A2E, in RPE cells. Thus the phenotype is remarkably similar between mice and that observed in the fundus of Stargardt patients.

The animal model and further biochemical investigations⁴⁹ suggest that ABCR functions as an outwardly directed flippase for N-retinylidene-PE. Delayed dark adaptation is likely due to accumulation (in discs) of the noncovalent complex between opsin and all-trans retinal. ABCR-mediated retinal degeneration in patients may result from “poisoning” of the RPE due to A2E accumulation, with secondary photoreceptor degeneration due to loss of the ABCR support role.

Though ABCR most definitely plays a role in the structure of discs, no mutations have been conclusively associated with abnormal structural features per se. In other words, it appears that mutations do not directly affect folding at the disc rim, and more likely affect the biochemical function of the transporter. In corroboration of these data, Radu and colleagues tested the effects of isotretinoin on lipofuscin accumulation in ABCR knockout mice,⁵⁰ a model of recessive Stargardt disease. They observed by electron microscopy that isotretinoin blocked both the formation of A2E biochemically, and the accumulation of lipofuscin pigments. Further, no significant visual loss was observed in ABCR null mice by electroretinography (when treated), and isotretinoin also blocked the slower, age-dependent accumulation of lipofuscin in wild-type mice. The results suggest that treatment with isotretinoin may inhibit lipofuscin accumulation and delay the onset of visual loss in patients with Stargardt disease and may be an effective treatment for other forms of retinal or macular degeneration associated with lipofuscin accumulation, though “normal” visual function may be somewhat compromised by such treatment.

The presence of an OS is clearly not optional, as there are many examples of retinal disease in which the photoreceptor cell dies shortly after loss of the OS.^51–53 Why loss of the OS triggers rod cell death is not obvious, since all of the required cellular organelles inhabit the IS and cell body; the OS appears devoid of organelles. The necessity to maintain an OS is further surprising since photoreceptors continually replace all the OS components. In fact, the renewal of rod cell OS components is perhaps more readily seen in the photoreceptor than in any other cell in the body. The disc and plasma membrane components of the OS are completely replaced within 2 weeks.^54,55 To support this turnover there is an influx of newly synthesized proteins and lipids from the IS. These components are transported to the base of the OS, and from there discs gradually migrate towards the RPE for phagocytosis and recycling. One idea is that the high oxygen tension coming from the choroidal vasculature may interfere with the cellular machinery in the IS and nucleus of the photoreceptor, and the presence of the OS puts a protective distance between the choroid and the photoreceptor nuclei.⁵⁶

Disc morphogenesis

Although it is known that new discs are formed at the base of the OS, the process by which these discs are formed is not fully resolved (Fig. 14.2). One model was described in the work of Anderson et al. (model 1).^57,58 Based on morphologic studies of adult monkey rods, it was observed that the folded membrane stacks at the base of the OS are continuous with the plasma membrane of the connecting cilium, whereas the older discs are closed off and separated from the plasma membrane. From this appearance a model was proposed that the newly arrived rhodopsin-bearing vesicles fuse with the plasma membrane, and the growing membrane evaginates to form open discs. Eventually these discs pinch off and form the mature closed discs. Another model (model 2), the vesicular targeting model, was recently proposed by Chuang and colleagues.⁵⁹ They provide molecular evidence that rhodopsin’s carboxyl terminus interacts with a protein called SARA (smad anchor for receptor activation), as well as phosphatidylinositol 3-phosphate (PI3P), and syntaxin 3. In their model, the new discs are already closed and enveloped by the OS plasma membrane. Their evidence suggests that the protein–protein and protein–lipid interactions organized by SARA regulate the vesicular targeting of axonemal rhodopsin-bearing vesicles to the newly formed discs. Thus the discs grow by fusing with the rhodopsin-bearing vesicles and not from the evaginated plasma membrane. This model is more consistent with the morphology of their experimental model system, the mouse rods, which do not display open discs at the base of the OS.

The degree of translational and posttranslational control in the processes of disc formation remains an open question. The processes necessary to manage and partition the correct ratio of rhodopsin to the other proteins of the phototransduction cascade (transducin: PDE: GC: GCAP: cGMP-gated channel) in the discs are unknown. Additionally, the insertion of the structural proteins peripherin, Rom-1, and ABCR into the forming disc rim is likely to require precise instruction. How these processes combine to generate a functional OS will require extensive new investigation and methodology. It is unlikely that these complex morphogenic processes will be elucidated using static two-dimensional microscopy methods with fixed materials, the method that has yielded most information to date. These problems may require the application of three-dimensional microscopy, in conjunction with immunocytochemical tags and fluorescent labels such as green fluorescent protein, to follow the assembling proteins in preparations of living photoreceptors.

Outer-segment plasma membrane

It is clear that, in addition to its obvious role in phototransduction, the OS plasma membrane contains a rich array of specialized proteins, many of which regulate the movement of ions into and out of the OS. The best-characterized proteins of this type are the retinal cGMP-gated (CNG) cation channels. In the dark-adapted rods and cones, Na⁺ and Ca²⁺ flow into the OS through these channels in the plasma membrane. Calcium comprises about 10% of the dark current carried by these channels in rods,⁶⁰ and perhaps 20% or more in cones.^61,62 In both rods and cones Na⁺ is extruded from the IS through the Na⁺/K⁺ pump. This flow of ions sets up the circulating dark current, of which the vast majority is carried by the Na⁺. The probability of the opening of the CNG channel, which in turn determines the size of the circulating current, depends on the amount of free [cGMP], which in the dark is estimated to be 3–4 µM.⁶³ At this concentration, the probability of channel opening is estimated to be only 0.1–0.2.^64,65 This underscores the impact that elevated [cGMP] can have on the number of open channels in the diseased state. The influx of Ca²⁺ through the channel is balanced by an efflux of Ca²⁺ by the Na⁺/Ca²⁺-K⁺ exchanger (NCKX) in the rod OS plasma membrane, thereby maintaining the intracellular level of Ca²⁺ at a relatively constant level.⁶⁶ Kaupp and colleagues⁶⁷ cloned the cGMP channel from bovine retina, while Pittler and colleagues⁶⁸ determined the primary structures of the human and mouse retinal rod cGMP-gated cation channel. These studies found that the sequence of the cGMP channel has significant similarity (59%) to the olfactory cAMP-gated channel. The retinal rod CNG channel is a hetero-oligomer composed of three alpha (CNGA1) and one beta (CNGB1) subunits,^69,70 each with cytoplasmic amino (N)- and carboxyl (C)-termini, six putative transmembrane domains, and a pore region.^71,72 Mutations in CNGA1 and CNGB1 have been linked to disease. A point mutation in CNGB1⁷³ and several mutations in CNGA1⁷⁴ have been identified and linked to recessive RP in humans. The CNG channel in cones consists of two CNGA3 and two CNGB3 subunits.⁷⁵ Together, mutations in either of these genes account for ~75% of complete acromatopsia.^76,77

In addition to the CNG channel, there are several other channels and transporters in the plasma membrane that serve to regulate the intracellular contents of the OS. The best studied of these is the NCKX. Rods express NCKX1 whereas cones express NCKX2.⁷⁸ The exchanger moves with every cycle 4 Na⁺ into the OS, and in exchange, moves one Ca²⁺ and one K⁺ into the subretinal space; exchange is thus electrogenic. Other transmembrane proteins, such as the GLUT-1 glucose transporter, have been shown to be present on both rod and cone OS.⁷⁹

Outer-segment lipids

Docosahexaenoic acid (DHA, 22:6n-6) is the major fatty acid found in the retinal rod OS, and rod photoreceptors have higher levels of DHA than in any other membrane system examined.⁸⁰ Numerous studies have established that the high level of DHA in rod OS membranes provides an optimal microenvironment for rhodopsin. DHA belongs to the n-3 family of essential polyunsaturated fatty acids. These fatty acids cannot be synthesized by vertebrates and they, or their shorter-chain precursors, must therefore be obtained in the diet.⁸¹

Humans with RP and dogs with progressive rod–cone degeneration (prcd) have lower than normal blood levels of long-chain polyunsaturated fatty acids, including DHA. In addition, prcd-affected dogs have lower levels of DHA in their rod OS than control animals.⁸² The reason for the reduced level of DHA in rod OS of animals with inherited retinal degeneration is not known. However, the fatty acid composition of the rod OS of these animals suggests that the synthesis of DHA-containing glycerolipids is downregulated in retinas of animals with inherited retinal degenerations.

Signal activation and amplification

A hallmark of rod phototransduction is its high sensitivity. Fully dark-adapted rods achieve sensitivity that reaches the theoretical maximum, the ability to detect individual quanta of light.^1,2 This high sensitivity is generated through several stages of amplification, resulting in a tremendous increase in the signal gain (Fig. 14.4). This cascade of amplification begins at the light-activated G-protein-coupled receptor rhodopsin (R). Absorption of a photon by R leads to a conformation change (R*), allowing it to interact with the heterotrimeric G-protein transducin (Tα, β, and γ), thereby promoting the exchange of bound GDP for GTP. The complex then dissociates into Tα-GTP and Tβγ, and R* is free to activate many other transducin molecules during its catalytic lifetime.⁸³ In the subsequent step, Tα-GTP binds and removes the inhibitory γ-subunit of the cGMP-PDE so that it can now hydrolyze cGMP to 5’-GMP; the enzymatic activity of PDE (PDE*) in turn degrades thousands of cGMP molecules. The decrease in cytoplasmic cGMP concentration then leads to closure of the cGMP-gated cation conductance channel in the rod plasma membrane, resulting in a reduction in the influx of ~ 1 000 000 Na⁺ ions.⁸⁴ This is a graded effect: a slight lowering in the cGMP concentration leads to closure of some channels, while a large decrease will eventually lead to closure of all channels. In this instance, the rod cell is said to be saturated.

Fig.14.4 (A) The phototransduction cascade in the rod and cone outer segment is initiated when a photon (hν) strikes the visual pigment, rhodopsin in rods, and promotes it to an active state (R*). R* interacts with a heterotrimeric G-protein (transducin) and promotes the exchange of guanosine triphosphate (GTP) for guanosine diphosphate (GDP) on the α-subunit (Tα). Tα in turn disinhibits a cyclic guanosine monophosphate (cGMP) phosphodiesterase (PDE), thereby allowing the hydrolysis of cGMP and the closure of cyclic nucleotide gated (CNG) channels. The closure of CNG channels interrupts the flow of Na⁺ and Ca²⁺ into the photoreceptor and hyperpolarizes the membrane potential. (B) Deactivation of R* is required to quench the phototransduction cascade. R* deactivation is initiated by the phosphorylation Ser and Thr residues at its C-terminus by rhodopsin kinase (RK). RK is inhibited by recoverin (Rv), a process which is controlled by Ca²⁺. Ca²⁺ concentration falls as CNG channels close during the activation of phototransduction, allowing the dissociation of RK from Rv, and subsequently the phosphorylation of R*. The R* catalytic activity is further quenched by the binding of visual arrestin (Arr) to phosphorylated R*. (C) Deactivation of activated PDE (PDE*) requires the hydrolysis of GTP to GDP on Tα. To reopen CNG channels, guanylyl cyclase synthesizes cGMP from GTP, a process that is regulated by the Ca²⁺-binding protein, guanylyl cyclase-activating protein 1 and 2 (GCAPs).

In darkness, glutamate, the transmitter used by both rods and cones, is released at a steady rate because the photoreceptor’s membrane potential is in a relatively depolarized state. The closure of CNG channels results in a graded hyperpolarization of the cell, leading to a decrease in glutamate release at the synapse. In this manner, the first sensation of light perception is transmitted from the photoreceptor cell to second-order cells of the retina, where signals are further processed and ultimately conveyed to the retinal ganglion cells (see Chapter 15, Functional anatomy of the mammalian retina).

Signal deactivation

A reversal of the activation steps is required ultimately for the photoreceptor to return to its resting state. First, the catalytically active components of the phototransduction cascade, R* and PDE*, must be quenched, then cGMP needs to be resynthesized (Fig. 14.4). These events must be rapid and reproducible for the photoreceptor to maintain its high sensitivity and respond to subsequent stimuli. Our current understanding of these processes is detailed below.

Deactivating PDE: control of transducin’s GTPase activity

Quenching of the phototransduction cascade also requires the shutoff of PDE*, which occurs through the deactivation of Tα. The GTPase activity of Tα allows the reassociation of the inhibitory γ-subunit to the catalytic PDE subunits,⁹⁶ a process which is speeded through the participation of a photoreceptor-specific RGS protein, RGS-9, and its binding partner, Gβ5.⁹⁷ These proteins act sequentially on Tα to facilitate the hydrolysis of bound GTP. The physiological role of all these proteins in the shutoff of the rod photoresponse was evaluated with transgenic mice that had alterations in each of these components. Rod photoresponses from transgenic mice expressing a PDE-γ with an impaired ability to stimulate GTP hydrolysis⁹⁸ showed abnormally slowed recovery, demonstrating the necessary role of PDE-γ in Tα deactivation.⁹⁹ Similarly, mice lacking RGS-9 showed profoundly slowed response recovery,¹⁰⁰ as do mice lacking Gβ5.¹⁰¹ RGS-9 is anchored to photoreceptor membranes by the adaptor protein, R9AP.^102–104 Interestingly, human patients with mutations in R9AP have difficulties adapting to sudden changes in light levels that are mediated by cones,¹⁰⁵ consistent with the higher concentration of RGS-9 complex in cones compared to rods.¹⁰⁶ Together, the current data support an important role of PDE-γ, RGS-9/Gβ5, and R9AP in Tα deactivation, and consequently, response recovery, in both rods and cones.

Light adaptation

In a normal cycle of day and night, the illumination at the Earth’s surface varies over 11 orders of magnitude, making photoreceptor adaptation fundamentally important to the normal functioning of the vertebrate visual system. Rods cells are able to adjust their sensitivity over 2–3 log units of light intensities, while cone cells exhibit no response saturation over 6–7 log units.^3,4 The switch between rod and cone function from night to bright daylight covers a large portion of vision’s functional range, and downstream circuitry accounts for the remainder of the range extension. While the molecular events underlying the amplification cascade following photon capture by rhodopsin are well delineated, the mechanisms underlying photoreceptor adaptation are less well understood; however, Ca²⁺ is known to play a feedback role in the adaptation process.^111,112

Adaptation mediated by Ca²⁺ feedback to retinal guanylyl cyclase

It has long been recognized that feedback controlling accelerated recovery contributes to light adaptation.¹⁰⁸ Recovery of the light response requires the resynthesis of cGMP, which is needed to reopen the CNG channels and re-establish the circulating current. The control of cGMP synthesis by RetGC/GCAPs is central to this process. The contribution of accelerated cGMP synthesis to adaptation in rod photoreceptors was investigated in GCAP knockout mice.^23,110 This work showed that the presence of GCAPs extends the sensitivity to background light in rod photoreceptors ~10-fold (Fig. 14.5), and can account for about half of the rod’s capacity to adapt to background light. Recordings from cones in GCAPs knockout mice also show that GCAP contributes to background adaptation, but to a lesser extent than it does in mouse rods.¹¹⁹

Fig. 14.5 Sensitivity as a function of background light was measured from mouse rod photoreceptors using suction electrodes (Fig. 14.3). Sensitivity was calculated from dim flash responses as the peak response amplitude divided by the flash strength. Flash sensitivity (S_F) in the presence of increasing background light, background light (I_B) was normalized against the flash sensitivity in darkness (S_F^D) and is plotted for the rods of several transgenic mice with particular elements of the phototransduction cascade altered. These mice include wild-type (•), mice lacking recoverin (), mice where the calmodulin-binding site on the cyclic nucleotide gated (CNG) channels has been eliminated (), mice lacking guanylyl cyclase-activating proteins 1 and 2 (), and mice where both the calmodulin-binding site on the CNG channel and guanylyl cyclase-activating proteins 1 and 2 have been eliminated (). The red line shows the predicted decline in sensitivity as a function of background light intensity if rods displayed no light adaptation. The blue line is best-fitting Weber–Fechner function for wild-type rods, and shows that light adaptation extends the sensitivity of rod photoreceptors to 100-fold higher background light levels. Rods from mice lacking recoverin, or the calmodulin-binding site on the CNG channels, show little deviation from wild-type rods, indicating that these proteins do not play a significant role in light adaptation. However, in mice lacking guanylyl cyclase-activating protein 1 and 2, light adaptation is impaired, but not absent, even when the calmodulin-binding site on the CNG channel is also eliminated (red dashed line). The remaining mechanisms that control light adaptation in rod photoreceptors remain unidentified.

(Data replotted from Chen J, Woodruff ML, Wang T, et al. Channel modulation and the mechanism of light adaptation in mouse rods. J Neurosci 2010;30:16232–40.)

Recoverin and control of rhodopsin kinase

The accelerated response recovery that mediates light adaptation is also achieved through the speeded deactivation of R*. The first step in the quenching of R* activity is the phosphorylation of its C-terminus by rhodopsin kinase, a process that is believed to be regulated in a Ca²⁺-dependent manner by the protein recoverin. Recoverin is a highly conserved Ca²⁺-binding protein found in both rod and cone photoreceptors. In vitro, recoverin binds and inhibits rhodopsin kinase when it is Ca²⁺-bound, and accelerates rhodopsin kinase activity as Ca²⁺ is removed.^120,121 Since [Ca²⁺]_i is high in darkness, it has been suggested that, under dark-adapted conditions, recoverin prolongs the catalytic activity of R* by inhibiting rhodopsin phosphorylation. Early evidence in experiments that introduce recombinant recoverin into gecko and salamander rods indicated that recoverin prolongs the light response.^122,123 However, recoverin’s effect was most prominent at Ca²⁺ concentrations outside the rod’s physiological range (>1 µM). The Ca²⁺ requirement for rhodopsin kinase inhibition by recoverin in reconstituted biochemical assays was similarly high.¹²⁰ These results placed into doubt the role of recoverin in mediating light adaptation. Suction electrode recordings from the rods of transgenic mice lacking recoverin, however, reveal a response speeding.¹²⁴ This speeding effect is exacerbated in rods that lack recoverin, but also have a reduced expression of rhodopsin kinase.¹²⁵ Thus, in the physiological range of Ca²⁺ concentrations, recoverin can influence R* lifetime. Despite this effect on R* lifetime, recoverin appears to play a relatively minor role in rod light adaptation (Fig. 14.5).¹²⁴ It remains to be seen in cone photoreceptors what is the efficacy of recoverin’s action. However, one might expect a greater contribution to adaptation as experiments from salamander L cones show that R* lifetime dominates the light response.¹²⁶

Feedback regulation of the cGMP-gated channel

Another target of the Ca²⁺ feedback is the CNG channel itself. In vitro data have shown that the presence of Ca²⁺ calmodulin causes a decrease in the apparent affinity of the CNG channels for cGMP.¹²⁷ This effect is reversed in light when [Ca²⁺]_i is low, such that more channels tend to open despite the lowering of cGMP levels. However, the role of CNG channel modulation in intact rods remains controversial, because, based upon the measured effect of Ca²⁺ on the apparent affinity of the channel for cGMP and the intracellular range of cGMP concentration, it has been predicted theoretically that the channel contributes little to the overall adaptive behavior of the rod photoreceptor.¹¹⁷ The site that confers Ca²⁺ calmodulin sensitivity has been identified in the β-subunit of the CNG channel.^128,129 Transgenic mice with this Ca²⁺-binding site disrupted show little influence on the light response or in the adaptive features of rod phototransduction,¹³⁰ consistent with previous conjecture (Fig. 14.5). The role of CNG channel modulation in cones, however, may be more profound than for rods. For instance, the cones of the ground squirrel demonstrate strong Ca²⁺-dependent modulation of the CNG-gated current in the normal range of Ca²⁺ concentrations, while the rods of the same species do not.¹³¹ In addition, Ca²⁺ concentration varies over a larger dynamic range in cones than rods.¹³² Modulation of cone CNG channels may also involve an unidentified soluble factor instead of calmodulin.¹³³ Ultimately how this modulation influences light adaptation in cones remains to be seen.

Other (Ca²⁺-independent) adaptation mechanisms: protein translocation

A mechanism operating at a longer timescale (minutes rather than seconds) that may contribute to regulating photoreceptor cell performance is the light-driven redistribution of specific signal transduction proteins between the compartments of the photoreceptor cell. The immunoreactivity of both Tα and visual arrestin is known to redistribute in rods in response to light.^134–137 Visual arrestin immunostaining predominates at rod IS, as well as at the outer nuclear layer and outer plexiform layers of dark-adapted retinas, and shifts to rod OS upon light exposure. Tα immunoreactivity shifts in the opposite direction in response to light.^136–138 Quantitative measurements show that bright light exposure is required to trigger translocation: for visual arrestin the threshold is 1000 rhodopsin excitations per rod per second, a light intensity at the upper limits of rod vision; for Tα at least 10 000 rhodopsin excitations are required.¹³⁹ Sokolov and colleagues confirmed the physiological movement of Tα by combining serial tangential cryosectioning of the retina with Western blot analysis, and demonstrated that such movement may help to extend the range of light intensities in which the rods are able to operate.¹⁴⁰ Similarly, visual arrestin movement may regulate rod performance under varying light environments.

Our current understanding of protein translocation is that it occurs by diffusion. In the case of Tα, the lipid modifications on the α- and γ-subunits act synergistically to anchor Tα GDP on the membrane. Upon activation, the subunits dissociate and become solubilized.¹⁴¹ In an elegant set of experiments, Lobanova and colleagues showed that light threshold can be shifted to either a lower or higher light intensity using mutant mice whose GTPase-activating (GAP) complex is enhanced or decreased.¹⁴² Their data suggest that, when the GAP complex is saturated, Tα GTP can escape the OS compartment and diffuse to the IS. Whether such a mechanism also governs cone translocation is uncertain. It has been observed that cones contain a higher concentration of GAP proteins,^106,143 a reason that could explain a much higher threshold for cone Tα translocation.¹⁴⁴ However, others have observed that cone Tα readily translocates.¹⁴⁵ In the case of rod arrestin, its interaction with microtubules restricts its location to the IS in the dark. Light exposure creates its high-affinity binding partner, R*, which drives its movement towards the OS.¹⁴⁶

It has become increasingly clear that Ca²⁺-dependent light adaptation results from the sum of multiple mechanisms that act at different sites in the phototransduction cascade. While biochemical experiments have identified the Ca²⁺-sensitive steps in the phototransduction cascade, a full understanding of how these steps contribute to visual adaptation requires a method that critically evaluates the timing, as well as the strength, of feedback modulation under physiological conditions. Given the complexity of Ca²⁺ feedback, coupled with the possibility that other adaptation mechanisms may overlay on this feedback regulation, visual adaptation, particularly in cones, remains one of the least understood areas in phototransduction. This complexity is being unraveled by the systematic removal of Ca²⁺ feedback pathways in vivo using mouse genetics, and by measuring the resultant change in adaptation with electrophysiological measurements. The molecular mechanism behind the observed phenotype can then be confirmed by biochemical experiments. Using this approach, the Ca²⁺ feedback loops can be evaluated independently, as well as in sum, since the mice can be bred to combine the individual components (Fig. 14.5). Eventually, as each and every Ca²⁺-dependent pathway is peeled away, the remaining (perhaps Ca²⁺-independent) pathways will be revealed.

Differences between rod and cone phototransduction

What factors underlie the specialized function of rods and cones? This remains a very important problem in phototransduction. Investigation of rod phototransduction has benefited from rod-dominated species where biochemical and physiological assays are robust. The relatively low number of cones in most mammalian species has made a biochemical analysis of cone phototransduction less fruitful, although the Nrl knockout mouse has proven useful for generating cone-dominant retinas.¹⁴⁷ As described above, some quantitative differences may arise for differential modulation of components of the phototransduction cascade. In addition, differences in their characteristic morphology may play a role in setting the response properties (i.e., the continuously invaginating OS plasma membrane in cones provides greater surface area for ion exchange and chromophore recycling). Also important may be their signal transduction cascades: molecular cloning has revealed that phototransduction in the vertebrate rods and cones is regulated by structurally homologous but distinct groups of signaling proteins. Therefore, it is reasonable to assume that the phototransduction in rods and cones is qualitatively similar, and that quantitative differences in the transduction steps underlie the characteristic rod and cone behavior. For example, R* may activate more rod Tα molecules than cone opsin R* in activating cone Tα, resulting in a higher gain and a larger response amplitude.

To determine the roles of rod and cone phototransduction proteins in setting the response properties of the photoreceptor cells, transgenic technology has recently been used to express rod components in cones, and vice versa. For example, Kefalov and colleagues¹⁴⁸ expressed an L-cone opsin in Xenopus rods, and rhodopsin in Xenopus cones. They surprisingly found that the activation of rhodopsin and L-cone opsin within the same cell yielded nearly identical photoresponses. In addition, similar photoresponses were observed in mouse rods transgenically expressing an S-cone pigment.¹⁴⁹ These results collectively rule out the visual pigment from being a major contributor to rod–cone functional differences. Similar experiments have also addressed the role of Tα. Deng and colleagues¹⁵⁰ showed no functional differences in mouse rods expressing cone Tα, or mouse cones expressing rod Tα. However, Chen and colleagues¹⁵¹ found to the contrary that complete exchange of rod Tα with cone Tα produced rod photoresponses that were 10-fold desensitized, and thus transducin might contribute partially to observed rod–cone functional differences. These results, as well as homolog substitution experiments with other components of phototransduction, require further evaluation to determine how differences in rod and cone photoresponses are established.

Inner segment

The functional role of the IS can be appreciated by examination of its unique anatomy, which is highly specialized to fuel the high energy and protein synthesis requirements of the photoreceptor. The metabolic functions of the IS are constantly and highly active, generating most of the rod cell’s energy as well as being the primary site for synthesizing new proteins. The proximal portion of the IS, termed the myoid, contains the endoplasmic reticulum and Golgi apparatus, while the distal portion, termed the ellipsoid, is densely packed with elongated mitochondria that are aligned circumferentially along the axis of the rod cell (Fig. 14.2). Cones have a larger IS, perhaps reflecting their higher metabolic needs. This photoreceptor cell compartment is structured for high O₂ consumption, as well as for glycolysis at very high rates. One major metabolic demand comes from the extrusion of Na⁺ from the cytoplasm by the Na⁺/K⁺ pump at the IS plasma membrane, which balances the influx of Na⁺ through the cGMP-gated channels at the OS. In darkness, these pumps operate at a very high rate to accommodate the high fluxes generated by many open channels in the OS, setting up the “dark” circulating current.¹⁵² The rate that these pumps can operate is related to the supply of ATP¹⁵³ generated by the mitochondria in the ellipsoid. This ATP synthesis rate is coupled with the O₂ delivery to the IS, which is completely supplied by the choroidal circulation.

Another major metabolic need is a high rate of protein synthesis that occurs in the myoid region of the IS to meet the demand of high levels of phototransduction proteins at the OS. In particular, each rod contains ~5 × 10⁷ rhodopsin molecules, of which 10% at the tip of the OS are phagocytosed by the RPE, beginning at the onset of light each day. To maintain homeostasis, ~5 × 10⁶ molecules are synthesized daily and added to the base of the OS. Similar events occur in cones, although they express about half the amount of visual pigment as rods. As with other cells, the soluble and peripheral membrane proteins are made in the cytosol, while the transmembrane proteins are made in the endoplasmic reticulum. To facilitate the speed of molecular collisions, many of the phototransduction proteins are associated with the membrane through lipid modifications and protein–protein interactions. These interactions are dynamic, with functional complexes forming and dissociating on the membrane surface on a rapid timescale. For example, the rod Tα is heterogeneously acylated at the amino-terminal glycine residue while the γ-subunit is carboxyl-methylated and prenylated at the cysteine residue. PDE6α and rhodopsin kinase are farnesylated, while PDE6β is geranylgeranylated.^154,155 In some instances, the lipid modification is exposed as a consequence of a change in protein structure. An example is recoverin that, upon binding calcium, exposes its amino-terminal myristoyl group.¹⁵⁶ GCAP1 and GCAP2 are also calcium-binding proteins that are myristoylated, but exposure of their lipid group is not regulated by calcium binding. These proteins are complexed with RetGCs and regulate their activity in a calcium-dependent manner.

The connecting cilium

A slender nonmotile connecting cilium that is 0.3 µm in diameter connects the IS to the OS. The microtubule-based axoneme of the connecting cilium lacks the central microtubule pair of the motile cilium, and therefore has a 9 × 2 + 0 microtubule structure towards the base of the cilia. The doublet microtubules transition to a 9 × 1 + 0 singlet microtubule arrangement more distally from the basal body and continue through much of the OS (Fig. 14.2). The axoneme arises from basal bodies which, together with the centrioles, act as microtubule organizing centers of the photoreceptor cell. It is the site of tremendous vectorial flow of lipids and proteins from their site of synthesis in the IS to the OS. It has been estimated that 2000 opsin molecules are transported every minute from the IS to the OS.¹⁷⁰ As post-Golgi carrier vesicles are delivered to the connecting cilium, proteins destined for the OS need to be sorted between the disc lamellar region, the disc rim region, and the plasma membrane in the OS. How the protein cargo is sorted at this point is not understood fully, but is thought to involve recognition of the specific trafficking motif displayed on the protein cargo by their cognate transport complex that also includes the intraflagellar transport (IFT) particles.¹⁷⁰ The IFT particles associate with the post-Golgi carrier vesicles at the base of the cilium and transport them along the connecting cilium to the OS. IFTs are large protein complexes that move along the axoneme by motor proteins and may be viewed as conveyer belts that deliver different cargos to their respective destination.

Actin and myosin, two proteins common to many cells in the body, are also present in photoreceptors. Both myosin and actin filaments are localized to the photoreceptor connecting cilium and IS/OS. Disruption of either of these proteins causes retinal degeneration.¹⁷¹ Actin depolymerization promotes disc overgrowth and photoreceptor death,¹⁷² and myosin mutants appear to have defects in the transport of molecules from the IS to the OS. Myosin defects cause numerous diseases, including Usher syndrome, that affect both hearing and vision.^173–175

Other proteins are receiving increased attention because of their association with the connecting cilium and human retinal disease. First described nearly two decades ago, one such protein, RP1, is responsible for 5–10% of all RP cases.¹⁷⁶ RP1 is a large protein localized to the connecting cilium in both rods and cones and is especially concentrated at the site of nascent OS discs. RP1 has recently been shown to bind microtubules, suggesting that this interaction may stabilize microtubules in the axoneme.^177–179 An RP1 knockout mouse model shows that discs are misshapen, overgrown, and fail to align properly.¹⁷⁷ This phenotype correlates well with ERG abnormalities in RP1 patients.

The RP GTPase regulator (RPGR) is a GTPase regulator found in rods. Mutations in RPGR¹⁸⁰ cause X-linked RP3, a severe and progressive retinal dystrophy. Antibodies localize RPGR and a related protein, RPGRIP (RPGR interacting protein) throughout the human rod and cone OS,¹⁸¹ as well as in amacrine cells. RPGR is found in connecting cilia of rods and cones and in the cilia of airway epithelia.¹⁸² An RPGR gene knockout mouse (an animal model of RP3)¹⁸³ demonstrated mislocalization of opsin in cones, and reduced quantities of rhodopsin in rods prior to photoreceptor degeneration. These data suggest a role for RPGR in maintaining the polarized distribution of proteins between the IS and the OS through the connecting cilium.

Centrins are calcium-binding proteins associated with centrosome-related structures. The mammalian rods and cones express four centrin isoforms; three of these (Cen1p–3p) are in the connecting cilium and three (Cen2p-4p) are in the basal body.¹⁸⁴ These centrins may participate in the alignment of the photoreceptor OS. Inasmuch as centrins were shown to interact with transducin in a calcium-dependent manner, they have been proposed to regulate the light-dependent translocation of Tα from the OS to the IS.¹⁸⁴

The ciliary rootlet, first recognized over a century ago, is a prominent structure originating from the basal body at the proximal end of a cilium. Rootlets appear to be particularly robust in retinal photoreceptors, extending from the basal bodies to the synaptic terminals, anchoring endoplasmic reticulum membranes along their length. Recent studies indicate that rootlets are composed of a structural protein, aptly named rootletin. Rootletin protofilaments are bundled into variably shaped thick filaments within the cilium.¹⁸⁵

Additional proteins, such as TULP1,^186,187 cadherin,¹⁸⁸ and myocilin,¹⁸⁹ are associated with the connecting cilium: several are known to be associated with rod photoreceptor disease. It suggests, not surprisingly, that the highly specialized connecting cilium is a complicated structure connecting the IS to OS, and that there will undoubtedly be additional proteins associated with this unique subcellular structure.

Nucleus

The nucleus contains the primary genome of the photoreceptor and is responsible for the initiation of genetic programs in the cell. As a result, the nucleus is the target for gene therapy protocols designed to correct genetic defects in the photoreceptor. Rods and cones differ in their nuclear features. The nucleus of the rod photoreceptor is rounder and is stained darker by nucleophilic stains due to the presence of a large clump of heterochromatin. Nuclei of cones are larger and oval in shape, with one to several clumps of heterochromatin and a larger amount of lightly staining euchromatin. Like most cells, photoreceptors also contain a second, nonnuclear genome in the mitochondria. Biochemical pathways linking photoreceptor cell death, the nucleus, and the mitochondria of the IS are beginning to come into focus.

Rhodopsin mutations

More than 100 different mutations of rhodopsin have been associated with autosomal dominant and recessive RP (adRP and arRP) and autosomal dominant congenital stationary night blindness.²¹³ This phenotypic heterogeneity points to diverse disease mechanisms arising from the same gene product. In the case of adRP, it has been proposed that some mutations lead to misfolding of the opsin protein, which then accumulates in the endoplasmic reticulum and elicits the unfolded protein response.²¹⁴ The accumulation of misfolded opsin may also interfere with the sorting and trafficking of normal proteins destined for the OS or other parts of the cell.²¹⁵ This may result in a pathologic mechanism where the cell cannot replenish other required proteins that do not harbor mutations.

Other rhodopsin mutations appear to affect trafficking of rhodopsin from the IS to the OS. As mentioned previously, mutations involving the last five carboxyl amino acids, such as P347A, P347S or S344ter, allow rhodopsin to appear to fold normally and to function normally in terms of Tα activation and deactivation by phosphorylation and visual arrestin binding. Yet, mutations in these residues cause adRP. Several lines of evidence suggest that the C-terminus of opsin contains a signal for vectorial transport of newly synthesized opsin toward the OS. In a retinal cell-free system, it was demonstrated that a monoclonal antibody to the C-terminal domain of rhodopsin inhibited post-Golgi vesicle formation and arrested newly synthesized rhodopsin in the trans-Golgi network.²¹⁶ A wild-type C-terminal peptide, but not a mutant peptide bearing the mutation at the P347 position, can also block this process.²¹⁷ Another line of evidence comes from an in vivo model where a truncation mutant, S344ter, was expressed in the photoreceptors of transgenic mice. Antibodies that specifically recognize the truncated opsin showed strong immunoreactivity in the IS, indicating a block in the transport to the OS.¹⁶³ However, some truncated opsins are also cotransported with the full-length protein to the OS. The role of the rhodopsin C-terminal domain in vectorial transport was also investigated in MDCK cells.²¹⁸ This cell line exhibits polarized structure when it is grown on a porous support. When transfected into this cell line, wild-type opsin was transported to the apical membrane surface, whereas an opsin mutant lacking the terminal 32 amino acids was not.

Constitutive phototransduction and retinal disease

It is thought that constitutive phototransduction causes some forms of retinal disorders. Unabated signal flow can arise from different steps in the visual cascade. For example, certain mutations in rhodopsin, particularly those that affect the salt bridge between Lys-296 and Glu-113 (such as A292E and G90D), can lead to constitutive activity.²¹⁹ The interaction between Lys-296 and Glu-113 constrains the chromophore-free opsin to an inactive conformation.²²⁰ Disruption of this bond leads to an opsin conformation that can support Tα activation. This activity is suppressed when the mutant opsin is regenerated with the chromophore 11-cis retinal. In the case of G90D and A292E mutations, light exposure leads to formation of opsin that can still activate the visual cascade. This can lead to impairment of visual function because the transduction machinery is overwhelmed by this constitutive activity. Patients carrying these mutations suffer from night blindness because phototransduction in rods cannot be fully quenched.^221,222 However, in the instance of the rhodopsin K296E mutation, no evidence of constitutive activity was observed when it was expressed in rod photoreceptors of transgenic mice; instead, it was found bound to arrestin.²²³ It was found subsequently that stable rhodopsin–arrestin complex is toxic, and rod photoreceptor survival can be prolonged if such complex is prevented in transgenic mice that lack visual arrestin and Tα.²²⁴

Defective shutoff can also be caused by the lack of visual arrestin or rhodopsin kinase. These genes are mutated in patients diagnosed with the recessive genetic disorder Oguchi disease,^225,226 a nonprogressive disorder characterized by early onset and stationary night blindness. These mutations are thought to cause a loss of function in the visual arrestin or rhodopsin kinase gene. Because of the inability to shut off signal flow, the rod photoreceptor becomes saturated even at very low light levels, leading to night blindness. Interestingly, some patients with the visual arrestin gene mutation also suffer from RP, pointing to a potential causal role between constitutive signal flow and photoreceptor cell death. Using the visual arrestin knockout mouse model, Chen et al. demonstrated that the absence of visual arrestin in the rod photoreceptor does lower the threshold for light damage.²²⁷ Therefore, protection from light exposure is an important preventive measure to prolong photoreceptor cell survival in those instances where constitutive signal flow is the disease mechanism.

Transducin defects and retinal disease

Transducin is composed of three subunits: α, β, and γ. Extensive genetic screening has revealed mutations only in the rod Tα.²²⁸ The associated disease is autosomal dominant congenital stationary night blindness. The mutation is in the position homologous to that affected by the oncogenic mutation in p21ras, a small G-protein. It is therefore hypothesized that this mutation may affect Tα deactivation, leading to the night blindness phenotype. Interestingly, mice that lack rod Tα have normal retinal structure, indicating that this abundant visual G-protein has no structural function in the photoreceptor, and that signal flow is not a prerequisite for rod photoreceptor cell survival.²⁰

cGMP and photoreceptor cell physiology

One of the first characterized mutations affecting components in the phototransduction cascade is the null mutation in the β-subunit of PDE6, leading to the rapid retinal degeneration phenotype in the rd mouse.^13,14 Subsequently, null mutations in PDE6β have been found in patients diagnosed with arRP.²²⁹ The complete PDE6 enzyme is made up of two catalytic chains (α and β), each associated with an inhibitory chain (γ). Since both α- and β-subunits are necessary for the formation of a functional PDE holoenzyme, the mutation in the γ-subunit renders the holoenzyme inactive. Farber and Lolley²³⁰ showed that photoreceptor cell death is preceded by an accumulation of cGMP in the retina, which is consistent with the role of this enzyme in degrading cGMP. Another study investigating the relationship between cGMP and photoreceptor cell death showed that external application of cGMP on to cultured eyecups resulted in rapid photoreceptor cell death.²³¹ However, the relevance of this finding is uncertain because the site of cGMP accumulation and action is intracellular. For example, the CNG channels open only when cGMP is dialyzed inside the truncated OS, not when it is applied externally.²³²

Although PDE6 is responsible for hydrolyzing cGMP, RetGCs are responsible for synthesizing the cGMP that is necessary for reopening of the CNG channel leading to recovery of the dark current. Two membrane forms of RetGC are expressed specifically in the retina of several mammalian species, including rat,²³³ human,^234,235 and bovine.²³⁶ Whereas other membrane cyclases are activated by the binding of peptide ligands to their extracellular domains, the activity of RetGCs is controlled by interaction with GCAPs through their intracellular domain.^237,238 Because the extracellular domain of RetGCs is situated in the enclosed and seemingly inaccessible intradiscal space, it is questionable whether the extracellular domain serves as a receptor. The retinal cyclases are therefore termed “orphan” receptors because no ligands have yet been identified. Although two forms of RetGC are expressed in the photoreceptor layer, there is evidence that they are not functionally redundant. First, they appear to be expressed at different levels in different photoreceptors. For example, it was observed that RetGC1 immunoreactivity is stronger in cone OS than in rod OS²³⁹; RetGC2, on the other hand, is expressed in the rod OS,²⁴⁰ but evidence for cone expression is lacking. Second, RetGC1 and RetGC2 behave differently biochemically in terms of their regulation by GCAPs: RetGC1 is regulated by both GCAP1 and GCAP2, whereas RetGC2 is regulated by GCAP2 but not by GCAP1.^{234,237–239,241} Third, loss of function of RetGC1 appears to affect cones more than rods; a mouse knockout of RetGC1 lacked cone function altogether, but rod function was only reduced.²⁴² The importance of RetGC1 in cones is also reflected in the rd chicken model where a loss of RetGC1 function is thought to cause retinal degeneration in this cone-dominant animal.²⁴³ These animal models will be valuable for evaluating the pathophysiology for Leber’s congenital amaurosis, an early-onset blindness caused by a naturally occurring mutation in RetGC1.

Another naturally occurring mutation that may influence intracellular cGMP concentration is the Y99C mutation in GCAP1, a mutation that is thought to cause cone dystrophy in an autosomal dominant manner.²⁴⁴ This mutation renders GCAP1 capable of stimulating RetGC, independently of Ca²⁺ concentration.^245,246 Constitutive activity of RetGC may therefore lead to an accumulation of intracellular cGMP.

It is known that a small increase in the level of free cGMP can profoundly affect the open probability of CNG channels, such that a greater number of channels will be open. This will in turn influence the ion influx, including Ca²⁺, which would then directly and/or indirectly influence the physiology of the photoreceptor cell, leading to cell death. In support of this idea, survival of rd1 photoreceptors, whose mutation in PDE6β leads to buildup of cGMP, was enhanced when the mutation was put into a CNG channel knockout mouse background.²⁴⁷ This observation motivates effort to synthesize small molecules that specifically block the CNG channel as a means of prolonging photoreceptor cell survival.²⁴⁸ A loss of RetGC1, on the other hand, is expected to lead to lowered cGMP levels. This might be functionally equivalent to constant light and may lead to cell death. However, further experiments will be needed to evaluate rigorously the relationship between cGMP concentration and retinal degeneration.