Structure and Function of Rod and Cone Photoreceptors

Transcription

1 PART 2 Basic Science and Translation to Therapy SECTION 1 Anatomy and Physiology Structure and Function of Rod and Cone Photoreceptors Jeannie Chen, Alapakkam P. Sampath Introduction Photoreceptor Fundamentals Photoreceptor Outer-Segment Structure Proteins That Stabilize the Structure of Outer-Segment Discs Disc Morphogenesis Outer-Segment Plasma Membrane Outer-Segment Lipids Phototransduction Signal Activation and Amplification Signal Deactivation Quenching R*: Rhodopsin Phosphorylation Quenching R*: Arrestin Binding Deactivating PDE: Control of Transducin s GTPase Activity Resynthesis of : Ca 2+ Dependence of Guanylyl Cyclase Light Adaptation The Role of Ca 2+ Feedback Adaptation Mediated by Ca 2+ -Feedback to Retinal Guanylyl Cyclase (RetGC) Recoverin and Control of Rhodopsin Kinase Feedback Regulation of the -Gated (CNG) Channel Other (Ca 2+ -Independent) Adaptation Mechanisms: Protein Translocation Dark Adaptation Differences Between Rod and Cone Phototransduction Inner Segment and Connecting Cilia Inner Segment Targeting of Phototransduction Proteins From the Inner Segment to the Outer Segment The Connecting Cilium Nucleus Photoreceptor Synaptic Terminal Photoreceptor Dysfunction and Disease Rhodopsin Mutations Constitutive Phototransduction and Retinal Disease Transducin Defects and Retinal Disease and Photoreceptor Cell Physiology INTRODUCTION Our visual experience is initiated by rod and cone photoreceptors in the retina. The human eye contains over 100 million rods and about 6 million cones, which are located within the outer nuclear layer of the retina and allow our visual experience to extend over 12 orders of magnitude in light intensity by splitting this range. Rod photoreceptors mediate vision under conditions of dim illumination, and allow our visual system to reach the limit imposed by the absorption of single photons. 1,2 Cones are less sensitive by ~100-fold, but their tremendous capacity for adaptation allows them to encode light intensities on the brightest of days. 3,4 Over the last two decades, our understanding of structure and function of these cells has increased dramatically. Over 150 genes have been cloned or linked to retinal diseases, and, surprisingly, as many as half of these genes are specifically expressed or highly enriched in the photoreceptor cells. Discovery of the molecular constituents of the rods and cones is progressing at an increasing rate, particularly enhanced by the availability of complete genomic sequences for both human and mouse. While photoreceptor genes can be identified through association with a retinal disease (linkage), information about their function does not accompany their identification. 5 Basic science research must then be undertaken to explain their role in both the normal, healthy photoreceptor, as well as in photoreceptor diseases. Though much of what we know about the structure and function of photoreceptor cells has come from studying animal models of inherited blindness, 6,7 more recently, transgenic and knockout animal technologies have established themselves as powerful tools for understanding function and studying disease. After new photoreceptor genes are identified from patients, mutant animals can be engineered to emulate human photoreceptor pathologies. Before these molecular technologies were available, most data about retinal disease was gleaned from the rare informative patient or donor retina that had surviving photoreceptors to examine. 8 Other major sources of information were studies of animal models that occurred through inbreeding or random inheritance of mutations in photoreceptor genes. Examples of these are the Irish setter dog, 9 the Briard dog, 10 the Abyssinian cat, 11 the RCS rat, 12 and the rd1 13,14 and rds 15, mouse models. Now, the ability to engineer transgenic animals has made the search for informative patients and naturally occurring animal models less acute since any single gene of interest can be introduced or removed from the photoreceptor. However, the most instructive examples of structure function relationships occur when there are patients and animal models with analogous mutation(s) and/or disease phenotype. Only recently has it become possible to incorporate the insights 387

2 388 PART 2 Basic Science and Translation to Therapy from numerous studies into biochemical pathways and mechanisms of photoreceptor function. Here, we review the principal proteins and pathways involved in rod photoreceptor structure and function, and highlight the parallelisms in cone photoreceptors. PHOTORECEPTOR FUNDAMENTALS The rod and cone photoreceptors are specialized sensory neurons that contain the protein machinery necessary to convert incident light into a signal that can be interpreted by the nervous system. Rod photoreceptors are more numerous than cones in most mammalian retina, and are highly sensitive. In the fully dark-adapted state, rods can reliably report the absorption of single photons to the retinal output, and they permit our scotopic, or night, vision. Cone photoreceptors are morphologically and functionally distinct from rods and express several types of visual pigments, or opsins, whose spectral sensitivity varies based on the cones subtype. In humans, three classes of cones confer robust color vision: S-cones, M-cones, and L-cones (see Chapter 12, Color vision and night vision). Cones are also less sensitive than rods and generate light responses that are temporally briefer. This allows cones to mediate our photopic, or day, vision with improved temporal resolution. The concerted action of these two types of photoreceptors, and the retinal circuitry that carries their signals to the retinal output, ultimately underlie our rich visual experience. Both rod and cone photoreceptors are highly polarized elongated cells that can be described as having four subcellular compartments: the outer segment (OS), the inner segment (IS), the nucleus, and the synaptic terminal (Figs..1 and.2). The OS is where photons are captured and activation of the phototransduction cascade begins. The IS lies immediately proximal to the OS, and contains the cell s protein synthesis (Golgi apparatus and endoplasmic reticulum) and metabolic (mitochondria) machinery. Light-evoked signals are relayed passively down the photoreceptor axon (up to 75 µm long) to the synaptic terminals in the outer plexiform layer. The structure of photoreceptor terminals is unique in the nervous system, as they contain a specialized structure called a ribbon that facilitates the release of the excitatory neurotransmitter glutamate onto second-order retinal neurons (bipolar and horizontal cells). Thus, the photoreceptor cells transduce the sensory stimulus, light, and pass on a signal to retinal circuits that carry this information to higher visual centers. PHOTORECEPTOR OUTER-SEGMENT STRUCTURE The OS compartment contains all components necessary for phototransduction, which is a set of biochemical reactions that convert photon capture to a change in a cationic current at the plasma membrane. Rods have a cylindrical OS about 1.3 µm in diameter; the OS length ranges from 25 to 45 µm, that depends on numerous factors including the time of day, the light intensity, their location in the retina, and the animal species. The cone OS is shorter, typically half the length as rods, with a larger diameter at the base that gradually tapers towards the tip (Fig..2). Mouse rod OS contains an average of 810 membranous discs, which occupy two-thirds of the volume of the OS. 29 The majority of the protein on these discs is rod opsin (or rhodopsin when the opsin is bound to its chromophore, 11-cis retinal). The density of rhodopsin on the disc membrane has been measured to be about 24,000 molecules/µm Despite this dense packing, rhodopsin freely diffuses in the membrane, 31 which facilitates its encounter with, and activation of, transducin molecules to amplify the light signal. Many G-protein-coupled receptors are known to form dimers and higher-order oligomers. Rhodopsin has been observed to form a paracrystalline array of dimers when native disc membrane is viewed under atomic force microscopy 32 and more recently by cryoelectron microscopy. 33 Whether it exists as dimers in vivo is still an area of controversy. However, it is known that rhodopsin is functional as monomers. 34 Other integral membrane proteins of the phototransduction pathway that are embedded in the discs are two isoforms of the guanylyl cyclases, RetGC1 and RetGC2, which are single-pass transmembrane proteins. Other transduction proteins, such as the G protein transducin (Tα), phosphodiesterase 6 (PDE6), recoverin, and guanylyl cyclase activating proteins (GCAPs), are peripheral membrane proteins (see below). 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 15,35,36 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. 37 Peripherin/rds is normally found within the edge or rim of the disc. It selfassociates to form higher-order complexes and also interacts with Rom 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. 39 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. 15,40 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. 15,40 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. 41 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, 37 and different peripherin/rds mutations appear to differentially affect rods and cones. For example, the C214S and N244K mutations have a greater effect on rods whereas R172W and N244H tend to cause conedominant diseases such as macular dystrophy. 37 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 A recent study of OS

3 Structure and Function of Rod and Cone Photoreceptors 389 A B C D E F Fig..1 (A) Scanning electron micrograph of rods and cones from the African clawed frog, Xenopus laevis. The basic shape of the rod photoreceptor inner and outer segments can be appreciated by this surface view. The cones in this species are exceptionally short and cone -shaped, unlike the cylindrical appearance of cone outer segment (OS) in the mammalian retina. (B) Immunolocalization of opsin in the inner segment (IS) of the frog retina. Arrows show location of newly synthesized opsin in the Golgi apparatus (solid arrows) and a track of labeled vesicles (open arrows) from the myoid to the cilium. (C) Transmission electron micrograph of photoreceptors from the mouse retina. Note the connecting cilium between the inner and outer segments and that a large number of mitochondria are visible in this orientation. (D) High-magnification view of frog photoreceptor OSs as viewed from the top of the disc stack. Upper left shows a single disc labeled with an anti-rod opsin antibody. The plasma membrane surrounding the disc can be observed in this section. Lower left is a cone photoreceptor OS that is not labeled by the antibody. (E) High-magnification view of frog photoreceptor OSs as viewed from the side of the disc stack. Discs are labeled with an anti-rod opsin antibody. The rim region of the disc can be observed, as well as the close packing of the individual discs within the OS. (F) Synaptic terminal of the rod photoreceptor. Directly below the nucleus is the photoreceptor spherule. Synaptic ribbons are visible in this section. (Panels D and E courtesy of Robert Molday, University of British Columbia.)

4 390 PART 2 Basic Science and Translation to Therapy Model 1 OS CC A BB Model 2 M G ER N S Rod Cone Fig..2 The fundamental organization of vertebrate rod and cone photoreceptors. Parts of the cell include: OS, outer segment; A, axoneme; CC, connecting cilium; BB, basal body; R, ciliary rootlet; M, mitochondria; G, Golgi apparatus; ER, endoplasmic reticulum; N, nucleus; S, synaptic terminal. Insets depict the structure of the axoneme at the level of the outer segment where microtubule structure is (top), and where the inner and outer segment join the microtubule structure is (bottom). Middle panels depict two models for outer segment (OS) disc formation. In Model 1, OS discs originate from evaginations of the plasma membrane but become closed off and separate from the plasma membrane. 56,57 In Model 2, newly generated OS discs are already closed and separate from the plasma membrane. 58 (Modified with permission from Anderson DH, Fisher SK, Steinberg RH. Mammalian cones: disc shedding, phagocytosis, and renewal. Invest Ophthalmol Vis Sci 1978;17(2):117-33; Steinberg RH, Fisher SK, Anderson DH. Disc morphogenesis in vertebrate photoreceptors. J Comp Neurol 1980;190(3):501-8; Chuang JZ, Zhao Y, Sung CH. SARA-regulated vesicular targeting underlies formation of the light-sensing organelle in mammalian rods. Cell 2007;130(3): ) structure using cryoelectron tomography of vitrified outer segments 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). 46,47 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. Other ABCR mutations are thought to increase the risk of developing age-related macular degeneration (AMD). ABCR is a member of the ATP-binding cassette (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

5 Structure and Function of Rod and Cone Photoreceptors 391 (N-retinylidene-PE), which is not found in normal retina. Once in the cytosol, the all-trans retinol moves to the RPE for recycling and chromophore (11-cis retinal) regeneration (see Chapter 18, 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 investigations 41 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 non-covalent 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, 49 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 wildtype 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 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 two weeks. 53,54 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. 55 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 (see Fig..2). One model was described in the work of Anderson, Steinberg, and Fisher (Model 1). 56,57 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, has been proposed by Chuang and colleagues. 58 They provide molecular evidence that rhodopsin s carboxyl terminus interacts with a protein called SARA (smad anchor for receptor activation), as well as PI3P (phosphatidylinositol 3-phosphate) 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 rhodopsinbearing 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: -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 of information to date. These problems may require the application of three-dimensional microscopy, in conjunction with immunocytochemical tags and fluorescent labels such as GFP, to follow the assembling proteins in preparations of living photoreceptors. Outer-Segment Plasma Membrane The plasma membrane of the rod OS encloses the majority of the discs and creates a physical barrier between the discs and the cell s exterior, except perhaps for the newly formed basal discs (see Fig..2, Model 1). In the mammalian cone OS at least several discs are continuous with the outer plasma membrane along the OS. 59 This open configuration creates additional surface area, which allows for more rapid exchange of retinoids and ions. 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 bestcharacterized proteins of this type are the retinal -gated (CNG) cation channels. In the dark-adapted rods and cones, Na + and Ca 2+ 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 [], 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 ,65 This underscores the impact that elevated [] can have on the number of open channels in the diseased state.

6 392 PART 2 Basic Science and Translation to Therapy The influx of Ca 2+ through the channel is balanced by an efflux of Ca 2+ by the Na + /Ca 2+ -K + exchanger in the rod OS plasma membrane, thereby maintaining the intracellular level of Ca 2+ at a relatively constant level. 66 Kaupp and colleagues 67 cloned the channel from bovine retina, while Pittler and colleagues 68 determined the primary structures of the human and mouse retinal rod -gated cation channel. These studies found that the sequence of the channel has significant similarity (59%) to the olfactory camp-gated channel. The retinal rod CNG channel is a hetero-oligomer composed of three alpha- (α) (CNGA1) and one beta- (β) (CNGB1) subunits, 69,70 each with cytoplasmic amino(n)- and carboxyl(c)- termini, six putative transmembrane domains, and a pore region. 15,71 Mutations in CNGA1 and CNGB1 have been linked to disease. A point mutation in CNGB1 72 and several mutations in CNGA1 73 have been identified and linked to recessive RP in humans. The CNG channel instead of cones consists of two CNGA3 and two CNGB3 subunits. 74 Together, mutations in either of these genes account for ~75% of complete acromatopsia. 75,76 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 Na + /Ca 2+ K + exchanger (NCKX), which is the only known route for Ca 2+ extrusion from the OS. Rods express NCKX1 whereas cones express NCKX2. 77 However, the lack of a clear phenotype in the cone electroretinogram in the NCKX2 knockout mice 78 raises possibilities for additional NCKX expression in cones or yet to be discovered NCKXindependent Ca 2+ extrusion mechanisms. The exchanger moves with every cycle 4 Na + into the OS, and in exchange, moves 1 Ca 2+ and 1 K + into the subretinal space; exchange is thus electrogenic. A mutation in NCKX1 is associated with autosomal recessive congenital stationary night blindness in humans, 79 and deletion of NCKX1 in mice resulted in malformed rod OS discs and suppressed expression and function of rod CNG channels. The rods slowly degenerated while cone function was maintained. 80 Other transmembrane proteins, such as the GLUT-1 glucose transporter, have been shown to be present on both rod and cone OS. 81 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. 82 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. 83 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. 84 The reason for the reduced level of DHA in rod outer segments 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. PHOTOTRANSDUCTION Visual transduction is initiated by the phototransduction cascade, which is perhaps the best characterized of all Fig..3 Suction electrode used in recording the outer-segment current. The cells in this clump of toad retina are being superfused with bicarbonate-buffered Ringer solution equilibrated with 5% carbon dioxide. A rod outer segment is carefully drawn into the glass electrode, which makes a high-resistance seal against the cell. Responses are recorded during the presentation of a stimulus, in this case a slit of focused green light. 1 (Modified with permission from Baylor DA, Lamb TD, Yau KW. Responses of retinal rods to single photons. J Physiol 1979;288: ) G-protein-coupled receptor signaling pathways. Our knowledge about phototransduction is relatively advanced because the components of this pathway are located selectively in the photoreceptor OS and can be isolated in quantities suitable for biochemical analysis. Photoreceptors can also be isolated easily for electrophysiologic measurements, either as single cells using suction electrodes (Fig..3), or en masse using field potentials called electroretinograms (ERG; see Chapter 9, Electrogenesis of the electroretinogram). The phototransduction cascade within rods is so robust such that single photon absorption by rhodopsin gives rise to a change in current that can be monitored by suction electrode recordings. Similarly, the remarkable ability of cone phototransduction to adapt to increases in background light intensity can also be monitored with suction electrodes. The combination of transgenic technology and electrophysiologic analysis has enhanced greatly our understanding of signal transduction within these photoreceptor cells. In particular, transgenic technologies have allowed the introduction of targeted changes into specific components of the signaling pathway. These genetically altered intact photoreceptor cells can then be subjected to electrophysiologic measurements, and their tissues can subsequently be used in biochemical or immunocytochemical assays to pinpoint the molecular mechanism behind the physiologic phenotype. Below, we summarize the current state of our understanding of phototransduction in retinal photoreceptors. 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..4). This cascade of amplification begins at the light-activated G-protein-coupled receptor rhodopsin

7 Structure and Function of Rod and Cone Photoreceptors 393 Intradiscal hv Rhodopsin R* Activation Phosphodiesterase Open Na + Ca 2+ A Cytosolic γ Ta β GDP γ Ta β GTP GDP Ta Ta β Ta a γ γ GTP GMP Closed Intradiscal R* Deactivation Closed R* RK RV RK P P P P P P B Ca 2+ Ca 2+ Cytosolic RV Arr Arr Closed Intradiscal PDE* Deactivation and Synthesis Open Guanylyl cyclase Ta a γ β γ Ta Ta γ Ta β + γ a β γ GCAPs GCAPs GTP Open Na + Ca 2+ C Cytosolic Fig..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 and promotes the exchange of GTP for GDP on the alpha-subunit (Tα). Tα in turn disinhibits a phosphodiesterase (PDE) thereby allowing the hydrolysis of and the closure of cyclic nucleotide gated (CNG) channels. The closure of CNG channels interrupts the flow of Na + and Ca 2+ 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 Ca 2+. Ca 2+ concentration falls as CNG channels close during the activation of phototransduction, allowing the disassociation 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 from GTP, a process that is regulated by the Ca 2+ -binding protein, guanylyl cyclase activating protein 1 and 2 (GCAPs). (R). Absorption of a photon by R leads to a conformation change (R*), allowing it to interact with the heterotrimeric G protein transducin (T alpha (α), beta (β), and gamma (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. 85 In the subsequent step, Tα-GTP binds and removes the inhibitory gamma- (γ) subunit of the -phosphodiesterase (PDE) so that it can now hydrolyze to 5 -GMP; the enzymatic activity of PDE (PDE*) in turn degrades thousands of molecules. The decrease in cytoplasmic concentration then leads to closure of the -gated cation conductance channel in the rod plasma membrane, resulting in a reduction in the influx of ~1,000,000 Na + ions. 86 This is a graded effect: a slight lowering in the concentration leads to closure of some channels, while

8 394 PART 2 Basic Science and Translation to Therapy a large decrease will eventually lead to closure of all channels. In this instance, the rod cell is said to be saturated. 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 secondorder cells of the retina, where signals are further processed and ultimately conveyed to the retinal ganglion cells (see Chapter 17, Function and 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 needs to be resynthesized (see Fig..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. Quenching R*: Rhodopsin Phosphorylation Since the early 1980s, phosphorylation was recognized to play an important role in deactivation of R*. 87,88 To determine how receptor deactivation occurs in vivo, transgenic mouse models were developed to assess the contribution of receptor phosphorylation to R* shut-off. It is clear that rhodopsin kinase, or GRK1, is solely responsible for phosphorylating R*, since phosphorylation of R* does not occur in mice lacking rhodopsin kinase. 89 In early experiments, a rhodopsin truncation mutation, S334ter, was expressed in the photoreceptors of transgenic mice 17 that removed the terminal 15 amino acid residues and thus all putative Ser and Thr phosphorylation sites. Single-photon responses produced by S334ter R* failed to shut off in a timely and stereotyped manner, indicating that this domain is important for R* quenching. In the 1990s numerous biochemical experiments suggested that specific Ser residues on the C-terminus are crucial in R* shut-off However, single cell recordings from transgenic mice rods expressing rhodopsin lacking native Ser or Thr residues showed that Ser-only rhodopsin generated abnormally prolonged responses whereas Thr-only rhodopsin generated responses that were only modestly slower than normal rhodopsin Quenching R*: Arrestin Binding Subsequent to C-terminus phosphorylation, the catalytic activity of R* is quenched fully through by binding of visual arrestin. Arrestins are soluble cytoplasmic proteins that bind to G-protein-coupled receptors, thus switching off activation of the G-protein and terminating the signaling pathway that triggers the cellular response; the most commonly studied arrestin is β-arrestin. Visual arrestin exhibits exquisite specificity for binding phosphorylated R*. Our understanding of how this specificity is conferred emerges from extensive mutagenesis studies, and the crystal structure of visual arrestin These studies reveal that arrestin is constrained into a latent, inactive structure by a network of intramolecular interactions. 96 According to the current model of arrestin activation, these intramolecular constraints are released by (1) interaction with multiple phosphates on rhodopsin s C-terminus, and (2) interaction with the cytoplasmic loops of R*. The physiologic role of visual arrestin in controlling the rod singlephoton response was studied by Xu and colleagues, 24 who created transgenic mice lacking visual arrestin. Suction electrode recordings of photoresponses from the OS of these transgenic rods displayed a rapid, partial recovery, followed by a prolonged final recovery phase. The timing of the slow phase of shutoff is consistent with the spontaneous decay of the R*. Thus, while R* phosphorylation is required to terminate rapidly its catalytic activity, the full quenching of R* activity requires visual arrestin binding. Together, these extensive studies of light responses arising from rhodopsin phosphorylation site mutants show that coordination of phosphorylation of the cluster of Ser and Thr sites, together with arrestin binding, play a pivotal role in controlling the catalytic lifetime of rhodopsin and in ensuring the reproducibility of the single photon response in rods. 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 gamma-subunit to the catalytic PDE subunits, 100 a process that is speeded through the participation of a photoreceptorspecific RGS protein, RGS-9, and its binding partner, Gβ These proteins act sequentially on Tα to facilitate the hydrolysis of bound GTP. The physiologic 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 hydrolysis 102 showed abnormally slowed recovery, demonstrating the necessary role of PDE-γ in Tα deactivation. 103 Similarly, mice lacking RGS-9 showed profoundly slowed response recovery, 104 as do mice lacking Gβ RGS-9 is anchored to photoreceptor membranes by the adaptor protein, R9AP Interestingly, human patients with mutations in R9AP have difficulties adapting to sudden changes in light levels that are mediated by cones, 109 consistent with the higher concentration of RGS-9 complex in cones compared to rods. 110 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. Resynthesis of : Ca 2+ Dependence of Guanylyl Cyclase Recovery of the dark current also requires that is resynthesized to allow CNG channels to open. is synthesized in photoreceptor outer segments by a retinal guanylyl cyclase (RetGC), which is regulated by the protein GCAPs (guanylyl cyclase activating protein 1 and 2). 111 GCAPs is an EF hand Ca 2+ -binding protein that confers on RetGC a highly cooperative Ca 2+ -dependence for synthesis. 112 Near the dark resting Ca 2+ concentration RetGC s activity is inhibited, but as OS Ca 2+ falls in response to illumination as CNG channels close, RetGC s activity accelerates to restore the resting and reopen CNG channels. The physiologic actions of GCAPs have been evaluated in physiologic recordings. For instance, when GCAP1 is dialyzed into Gecko rods, the recovery phase of the light response is accelerated. 113 In addition, suction electrode recordings from rods of GCAPs knockout mice show that the Ca 2+ -dependence of synthesis is required to set the time course of the rod s photoresponse, and to suppress noise within the phototransduction cascade. 114 Light Adaptation In a normal cycle of day and night, the illumination at the earth s surface varies over 12 orders of magnitude, making

9 Structure and Function of Rod and Cone Photoreceptors 395 photoreceptor adaptation fundamentally important to the normal functioning of the vertebrate visual system. Rod cells are able to adjust their sensitivity over 2 to 3 log units of light intensities, while cone cells exhibit no response saturation over 6 to 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, Ca 2+ is known to play a feedback role in the adaptation process. 105,115 The Role of Ca 2+ Feedback In darkness, Na + ions enter the OS through the CNG channels and are extruded in the IS through Na + /K + ATPase pumps. This steady-state influx and efflux of Na + is largely the basis for the circulating dark current in photoreceptor cells. Similarly, Ca 2+ enters through CNG channels in the OS, but is extruded locally by Na + /Ca 2+, K + (NCKX) exchangers. In mouse rods the steady-state influx and efflux of Ca 2+ holds its concentration near 250 nm, 1 but as light closes CNG channels Ca 2+ entry is blocked, Ca 2+ efflux continues, and [Ca 2+ ] i falls. This change in [Ca 2+ ] i is graded just like the dark current: the degree of lowered [Ca 2+ ] i is proportional to the intensity of background light in both rods and cones. 117,118 This decrease in [Ca 2+ ] i triggers a feedback signal that is necessary for light adaptation, since, when the [Ca 2+ ] i decrease is blocked, adaptation to background illumination is greatly compromised. 60,119 Existing evidence suggests that this feedback is rather complex, as it orchestrates several pathways directed toward different components of the transduction machinery Here we review the role of Ca 2+ on each of these components. Adaptation Mediated by Ca 2+ -Feedback to Retinal Guanylyl Cyclase (RetGC) It has long been recognized that feedback controlling accelerated recovery contributes to light adaptation. 112 Recovery of the light response requires the resynthesis of, which is needed to reopen the CNG channels and reestablish the circulating current. The control of synthesis by RetGC/ GCAPs is central to this process. The contribution of accelerated synthesis to adaptation in rod photoreceptors was investigated in GCAPs knockout mice. 23,114 This work showed that the presence of GCAPs extends the sensitivity to background light in rod photoreceptors ~10-fold (Fig..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 GCAPs contributes to background adaptation, but to a lesser extent than it does in mouse rods. 123 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 Ca 2+ -dependent manner by the protein recoverin. Recoverin is a highly conserved Ca 2+ - binding protein found in both rod and cone photoreceptors. In vitro, recoverin binds and inhibits rhodopsin kinase when it is Ca 2+ -bound, and accelerates rhodopsin kinase activity as Ca 2+ is removed. 124,125 Since [Ca 2+ ] 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 SF/SF D I B (photons µm 2 s 1 ) Fig..5 Sensitivity as a function of background light was measured from mouse rod photoreceptors using suction electrodes (see Fig..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 (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 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 dashed line shows the predicted decline in sensitivity as a function of background light intensity if rods displayed no light adaptation. The solid 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 CK, Woodruff ML, Chen FS, Chen D, Fain GL. Background light produces a recoverin-dependent modulation of activated-rhodopsin lifetime in mouse rods. J Neurosci 2010;30(4): ) that introduce recombinant recoverin into Gecko and salamander rods indicated that recoverin prolongs the light response. 126,127 However, recoverin s effect was most prominent at Ca 2+ concentrations outside the rod s physiologic range (>1 µm). The Ca 2+ requirement for rhodopsin kinase inhibition by recoverin in reconstituted biochemical assays was similarly high. 124 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. 128 This speeding effect is exacerbated in rods that lack recoverin, but also have a reduced expression of rhodopsin kinase. 129 Thus, in the physiologic range of Ca 2+ 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..5). 128 In cone photoreceptors, however, recoverin appears to contribute to sensitivity over a wider range of light intensities, 130 a fact perhaps not totally surprising given experiments

10 396 PART 2 Basic Science and Translation to Therapy from salamander L cones show that R* lifetime dominates the light response. 131 Feedback Regulation of the -Gated (CNG) Channel Another target of the Ca 2+ feedback is the CNG channel itself. In vitro data have shown that the presence of Ca 2+ -calmodulin causes a decrease in the apparent affinity of the CNG channels for. 132 This effect is reversed in light when [Ca 2+ ] i is low, such that more channels tend to open despite the lowering of levels. However, the role of CNG channel modulation in intact rods remains controversial, because based upon the measured effect of Ca 2+ on the apparent affinity of the channel for and the intracellular range of concentration, it has been predicted theoretically that the channel contributes little to the overall adaptive behavior of the rod photoreceptor. 121 The site that confers Ca 2+ -calmodulin sensitivity has been identified in the beta-subunit of the CNG channel. 133,134 Transgenic mice with this Ca 2+ -binding site disrupted show little influence on the light response or in the adaptive features of rod phototransduction, 129 consistent with previous conjecture (see Fig..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 Ca 2+ -dependent modulation of the CNG gated-current in the normal range of Ca 2+ concentrations, while the rods of the same species do not. 135 In addition, Ca 2+ concentration varies over a larger dynamic range in cones than rods 118 allowing the recently identified protein, CNG-modulin, to regulate cone sensitivity more profoundly. 136,137 Other (Ca 2+ -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 Visual arrestin immunostaining predominates at rod ISs, as well as at the outer nuclear layer and outer plexiform layers of dark-adapted retinas, and shifts to rod OSs upon light exposure. Tα immunoreactivity shifts in the opposite direction in response to light 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 is required. 143 Sokolov and colleagues confirmed the physiologic 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. 144 More recent evidence indicates that movement of Tα to synaptic terminal enhances synaptic transmission in the light-adapted state. 145 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 alpha- and gamma-subunits act synergistically to anchor Tα-GDP on the membrane. Upon activation, the subunits dissociate and become solubilized. 146 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. 147 Their data suggests 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, 110,148 a reason that could explain a much higher threshold for cone Tα translocation. 149 However, others have observed that cone Tα readily translocates. 150 In the case of rod arrestin, its interaction with microtubules restricts its location to the IS in the dark. Light exposure creates its highaffinity binding partner, R*, which drives its movement towards the OS. 151 It has become increasingly clear that Ca 2+ -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 Ca 2+ sensitive steps in the phototransduction cascade, a full understanding of how these steps contributes to visual adaptation requires a method that critically evaluates the timing, as well as the strength, of feedback modulation under physiologic conditions. Given the complexity of Ca 2+ 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 Ca 2+ feedback pathways in vivo using mouse genetics, and by measuring the resultant change in adaptation with electrophysiologic measurements. The molecular mechanism behind the observed phenotype can then be confirmed by biochemical experiments. Using this approach, the Ca 2+ feedback loops can be evaluated independently, as well as in sum, since the mice can be bred to combine the individual components (see Fig..5). Eventually, as each and every Ca 2+ -dependent pathway is peeled away, the remaining (perhaps Ca 2+ -independent) pathways will be revealed. Dark Adaptation The process of light adaptation described above represents the mechanisms that desensitize phototransduction during steady background light. Despite the push toward desensitization by these mechanisms, several of which depend on the ROS Ca 2+ concentration, there is a simultaneous pull toward sensitization. Adaptive mechanisms that pull the system toward the dark state (collectively coined dark adaptation ) play a crucial role in sensitivity maintenance. Dark adaptation has been classically studied by exposing dark-adapted subjects, or animal retinas, to bright light that bleaches (i.e., causes a cis to trans isomerization of retinal) the visual pigment and depresses sensitivity, and then observing the recovery with time. While cones recover sensitivity within seconds to minutes, rods take tens of minutes to recover to levels comparable to the dark-adapted state. 152 This slow recovery is attributable to many biochemical processes within rods that quench phototransduction activity fully and reestablish the darkadapted condition. Among these is the rapid and continuous turnover of the visual pigment, which in rods requires tight apposition with the RPE. Additionally, mechanisms that reset the distribution and state of phototransduction proteins are critical to establish sensitivity. These mechanisms include the decay of long-lasting visual pigment intermediates, such as MetaIII rhodopsin (reviewed in reference 153), and reversing the light-dependent movement of proteins such as Tα, recoverin, and arrestin back to their respective compartments (reviewed in reference 143). The clinical importance of dark adaptation is manifest in pathologic perturbations of this process that lead to a number of blinding eye diseases, including retinal degeneration and diseases of retinoid deficiency.