RESUMO
Capture of a photon by an opsin visual pigment isomerizes its 11-cis-retinaldehyde (11cRAL) chromophore to all-trans-retinaldehyde (atRAL), which subsequently dissociates. To restore light sensitivity, the unliganded apo-opsin combines with another 11cRAL to make a new visual pigment. Two enzyme pathways supply chromophore to photoreceptors. The canonical visual cycle in retinal pigment epithelial cells supplies 11cRAL at low rates. The photic visual cycle in Müller cells supplies cones with 11-cis-retinol (11cROL) chromophore precursor at high rates. Although rods can only use 11cRAL to regenerate rhodopsin, cones can use 11cRAL or 11cROL to regenerate cone visual pigments. We performed a screen in zebrafish retinas and identified ZCRDH as a candidate for the enzyme that converts 11cROL to 11cRAL in cone inner segments. Retinoid analysis of eyes from Zcrdh-mutant zebrafish showed reduced 11cRAL and increased 11cROL levels, suggesting impaired conversion of 11cROL to 11cRAL. By microspectrophotometry, isolated Zcrdh-mutant cones lost the capacity to regenerate visual pigments from 11cROL. ZCRDH therefore possesses all predicted properties of the cone 11cROL dehydrogenase. The human protein most similar to ZCRDH is RDH12. By immunocytochemistry, ZCRDH was abundantly present in cone inner segments, similar to the reported distribution of RDH12. Finally, RDH12 was the only mammalian candidate protein to exhibit 11cROL-oxidase catalytic activity. These observations suggest that RDH12 in mammals is the functional ortholog of ZCRDH, which allows cones, but not rods, to regenerate visual pigments from 11cROL provided by Müller cells. This capacity permits cones to escape competition from rods for visual chromophore in daylight-exposed retinas.
Assuntos
Oxirredutases do Álcool , Células Fotorreceptoras Retinianas Cones , Células Fotorreceptoras Retinianas Bastonetes , Peixe-Zebra , Animais , Peixe-Zebra/fisiologia , Células Fotorreceptoras Retinianas Cones/metabolismo , Células Fotorreceptoras Retinianas Cones/fisiologia , Células Fotorreceptoras Retinianas Bastonetes/metabolismo , Células Fotorreceptoras Retinianas Bastonetes/fisiologia , Oxirredutases do Álcool/metabolismo , Oxirredutases do Álcool/genética , Proteínas de Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/genética , Retinaldeído/metabolismo , Pigmentos da Retina/metabolismo , Humanos , Opsinas/metabolismo , Opsinas/genéticaRESUMO
Though rod and cone photoreceptors use similar phototransduction mechanisms, previous model calculations have indicated that the most important differences in their light responses are likely to be differences in amplification of the G-protein cascade, different decay rates of phosphodiesterase (PDE) and pigment phosphorylation, and different rates of turnover of cGMP in darkness. To test this hypothesis, we constructed TrUx;GapOx rods by crossing mice with decreased transduction gain from decreased transducin expression, with mice displaying an increased rate of PDE decay from increased expression of GTPase-activating proteins (GAPs). These two manipulations brought the sensitivity of TrUx;GapOx rods to within a factor of 2 of WT cone sensitivity, after correcting for outer-segment dimensions. These alterations did not, however, change photoreceptor adaptation: rods continued to show increment saturation though at a higher background intensity. These experiments confirm model calculations that rod responses can mimic some (though not all) of the features of cone responses after only a few changes in the properties of transduction proteins.
Assuntos
Células Fotorreceptoras Retinianas Cones , Células Fotorreceptoras Retinianas Bastonetes , Transducina , Animais , Células Fotorreceptoras Retinianas Cones/metabolismo , Células Fotorreceptoras Retinianas Bastonetes/metabolismo , Camundongos , Transducina/metabolismo , Transducina/genética , Retina/metabolismo , Diester Fosfórico Hidrolases/metabolismo , Diester Fosfórico Hidrolases/genéticaRESUMO
Mammalian UNC119 is a ciliary trafficking chaperone highly expressed in the inner segment of retinal photoreceptors. Previous research has shown that UNC119 can bind to transducin, the synaptic ribbon protein RIBEYE, and the calcium-binding protein CaBP4, suggesting that UNC119 may have a role in synaptic transmission. We made patch-clamp recordings from retinal slices in mice with the UNC119 gene deleted and showed that removal of even one gene of UNC119 has no effect on the rod outer segment photocurrent, but acted on bipolar cells much like background light: it depolarized membrane potential, decreased sensitivity, accelerated response decay, and decreased the Hill coefficient of the response-intensity relationship. Similar effects were seen on rod bipolar-cell current and voltage responses, and after exposure to bright light to translocate transducin into the rod inner segment. These findings indicate that UNC119 deletion reduces the steady-state glutamate release rate at rod synapses, though no change in the voltage dependence of the synaptic Ca current was detected. We conclude that UNC119, either by itself or together with transducin, can facilitate the release of glutamate at rod synapses, probably by some interaction with RIBEYE or other synaptic proteins rather than by binding to CaBP4 or calcium channels.
Assuntos
Transmissão Sináptica , Transducina , Animais , Camundongos , Glutamatos/metabolismo , Mamíferos/metabolismo , Retina/metabolismo , Sinapses/metabolismo , Transmissão Sináptica/fisiologia , Transducina/metabolismoRESUMO
Most defects causing retinal degeneration in retinitis pigmentosa (RP) are rod-specific mutations, but the subsequent degeneration of cones, which produces loss of daylight vision and high-acuity perception, is the most debilitating feature of the disease. To understand better why cones degenerate and how cone vision might be restored, we have made the first single-cell recordings of light responses from degenerating cones and retinal interneurons after most rods have died and cones have lost their outer-segment disk membranes and synaptic pedicles. We show that degenerating cones have functional cyclic-nucleotide-gated channels and can continue to give light responses, apparently produced by opsin localized either to small areas of organized membrane near the ciliary axoneme or distributed throughout the inner segment. Light responses of second-order horizontal and bipolar cells are less sensitive but otherwise resemble those of normal retina. Furthermore, retinal output as reflected in responses of ganglion cells is less sensitive but maintains spatiotemporal receptive fields at cone-mediated light levels. Together, these findings show that cones and their retinal pathways can remain functional even as degeneration is progressing, an encouraging result for future research aimed at enhancing the light sensitivity of residual cones to restore vision in patients with genetically inherited retinal degeneration.
Assuntos
Visão de Cores , Degeneração Retiniana , Retinose Pigmentar , Humanos , Degeneração Retiniana/metabolismo , Células Fotorreceptoras Retinianas Cones/fisiologia , Retina/metabolismo , Retinose Pigmentar/genética , Retinose Pigmentar/metabolismoRESUMO
Mice detect decreases in illumination in dim light near the visual threshold with OFF retinal ganglion cells.
Assuntos
Visão Ocular , Percepção Visual , Animais , Iluminação , Camundongos , Estimulação Luminosa , Células Ganglionares da RetinaRESUMO
Retinal bipolar cells receive direct input from rod and cone photoreceptors and send axons into the inner retina, synapsing onto amacrine and ganglion cells. Bipolar cell responses can be either depolarizing (ON) or hyperpolarizing (OFF); in lower vertebrates, bipolar cells receive mixed rod and cone input, whereas in mammals, input is mostly segregated into 14 classes of cone ON and OFF cells and a single rod ON bipolar cell. We show that lamprey, like mammals, have rod bipolar cells with little or no cone input, but these cells are OFF rather than ON. They have a characteristic morphology and a spectral sensitivity nearly indistinguishable from that of rod photoreceptors. In background light known to saturate rods, rod bipolar cells are also saturated and cannot respond to increment flashes. Our results suggest that early vertebrate progenitors of both agnathans and gnathostomes may have had a more fluid retinal organization than previously thought.
Assuntos
Petromyzon , Células Bipolares da Retina , Animais , Mamíferos , Retina/fisiologia , Células Fotorreceptoras Retinianas Cones/fisiologia , Células Fotorreceptoras Retinianas Bastonetes , VertebradosRESUMO
The high sensitivity of night vision requires that rod photoreceptors reliably and reproducibly signal the absorption of single photons, a process that depends on tight regulation of intracellular cGMP concentration through the phototransduction cascade. Here in the mouse (Mus musculus), we studied a single-site D167A mutation of the gene for the α subunit of rod photoreceptor phosphodiesterase (PDEA), made with the aim of removing a noncatalytic binding site for cGMP. This mutation unexpectedly eliminated nearly all PDEA expression and reduced expression of the ß subunit (PDEB) to â¼5%-10% of WT. The remaining PDE had nearly normal specific activity; degeneration was slow, with 50%-60% of rods remaining after 6 months. Responses were larger and more sensitive than normal but slower in rise and decay, probably from slower dark turnover of cGMP. Remarkably, responses became much less reproducible than WT, with response variance increasing for amplitude by over 10-fold, and for latency and time-to-peak by >100-fold. We hypothesize that the increase in variance is the result of greater variability in the dark-resting concentration of cGMP, produced by spatial and temporal nonuniformity in spontaneous PDE activity. This variability decreased as stimuli were made brighter, presumably because of greater spatial uniformity of phototransduction and the approach to saturation. We conclude that the constancy of the rod response depends critically on PDE expression to maintain adequate spontaneous PDE activity, so that the concentration of second messenger is relatively uniform throughout the outer segment.SIGNIFICANCE STATEMENT Rod photoreceptors in the vertebrate retina reliably signal the absorption of single photons of light by generating responses that are remarkably reproducible in amplitude and waveform. We show that this reproducibility depends critically on the concentration of the effector enzyme phosphodiesterase (PDE), which metabolizes the second messenger cGMP and generates rod light responses. In rods with the D167A mutation of the α subunit of PDE, only 5%-10% of PDE is expressed. Single-photon responses then become much more variable than in WT rods. We think this variability is caused by spatial and temporal inhomogeneity in the concentration of cGMP in darkness, so that photons absorbed in different parts of the cell produce responses of greatly varying amplitude and waveform.
Assuntos
GMP Cíclico , Diester Fosfórico Hidrolases , Animais , GMP Cíclico/metabolismo , Camundongos , Diester Fosfórico Hidrolases/genética , Diester Fosfórico Hidrolases/metabolismo , Reprodutibilidade dos Testes , Retina/metabolismo , Células Fotorreceptoras Retinianas Bastonetes/metabolismoRESUMO
Cone photoreceptors provide the foundation of most of human visual experience, but because they are smaller and less numerous than rods in most mammalian retinas, much less is known about their physiology. We describe new techniques and approaches which are helping to provide a better understanding of cone function. We focus on several outstanding issues, including the identification of the features of the phototransduction cascade that are responsible for the more rapid kinetics and decreased sensitivity of the cone response, the roles of inner-segment voltage-gated and Ca2+-activated channels, the means by which cones remain responsive even in the brightest illumination, mechanisms of cone visual pigment regeneration in constant light, and energy consumption of cones in comparison to that of rods.
Assuntos
Estimulação Luminosa , Células Fotorreceptoras Retinianas Cones/metabolismo , Visão Ocular/fisiologia , Animais , Cálcio/metabolismo , Humanos , Estimulação Luminosa/métodosRESUMO
Rod photoreceptors can be saturated by exposure to bright background light, so that no flash superimposed on the background can elicit a detectable response. This phenomenon, called increment saturation, was first demonstrated psychophysically by Aguilar and Stiles and has since been shown in many studies to occur in single rods. Recent experiments indicate, however, that rods may be able to avoid saturation under some conditions of illumination. We now show in ex vivo electroretinogram and single-cell recordings that in continuous and prolonged exposure even to very bright light, the rods of mice from both sexes recover as much as 15% of their dark current and that responses can persist for hours. In parallel to recovery of outer segment current is an â¼10-fold increase in the sensitivity of rod photoresponses. This recovery is decreased in transgenic mice with reduced light-dependent translocation of the G protein transducin. The reduction in outer-segment transducin together with a novel mechanism of visual-pigment regeneration within the rod itself enable rods to remain responsive over the whole of the physiological range of vision. In this way, rods are able to avoid an extended period of transduction channel closure, which is known to cause photoreceptor degeneration.SIGNIFICANCE STATEMENT Rods are initially saturated in bright light so that no flash superimposed on the background can elicit a detectable response. Frederiksen and colleagues show in whole retina and single-cell recordings that, if the background light is prolonged, rods slowly recover and can continue to produce significant responses over the entire physiological range of vision. Response recovery occurs by translocation of the G protein transducin from the rod outer to the inner segment, together with a novel mechanism of visual-pigment regeneration within the rod itself. Avoidance of saturation in bright light may be one of the principal mechanisms the retina uses to keep rod outer-segment channels from ever closing for too long a time, which is known to produce photoreceptor degeneration.
Assuntos
Células Fotorreceptoras Retinianas Bastonetes/metabolismo , Transducina/metabolismo , Animais , Eletrorretinografia , Feminino , Luz , Masculino , Camundongos , Transporte Proteico , Células Fotorreceptoras Retinianas Bastonetes/fisiologia , Células Fotorreceptoras Retinianas Bastonetes/efeitos da radiação , Análise de Célula Única , Transducina/genética , Visão OcularRESUMO
The discoveries of the photopigment melanopsin and intrinsically photosensitive retinal ganglion cells (ipRGCs) have revealed novel mechanisms of light detection now known to control several kinds of non-image-forming vision, including regulation of mood, the circadian rhythm, and the pupillary light reflex (PLR). These remarkable discoveries have been made mostly on mammals, but many vertebrates express melanopsin and adjust the diameter of the pupil to the ambient light intensity to extend the operating range of vision and reduce spherical aberration1. We were curious to know whether a PLR controlled by melanopsin is also present in lamprey, which are members of the only remaining group of jawless vertebrates (agnathans) which diverged from all other vertebrates about 500 million years ago2. We now show that lamprey have a robust PLR mediated by melanopsin apparently without any contribution from signals of rods and cones, suggesting that non-image-forming perception emerged long before the radiation of present vertebrate lines and was already present in the late Cambrian.
Assuntos
Petromyzon/fisiologia , Reflexo Pupilar/fisiologia , Visão Ocular/fisiologia , Animais , Células Ganglionares da Retina/metabolismo , Células Horizontais da Retina/metabolismo , Opsinas de Bastonetes/metabolismoRESUMO
We have used recent measurements of mammalian cone light responses and voltage-gated currents to calculate cone ATP utilization and compare it to that of rods. The largest expenditure of ATP results from ion transport, particularly from removal of Na+ entering outer segment light-dependent channels and inner segment hyperpolarization-activated cyclic nucleotide-gated channels, and from ATP-dependent pumping of Ca2+ entering voltage-gated channels at the synaptic terminal. Single cones expend nearly twice as much energy as single rods in darkness, largely because they make more synapses with second-order retinal cells and thus must extrude more Ca2+ In daylight, cone ATP utilization per cell remains high because cones never remain saturated and must continue to export Na+ and synaptic Ca2+ even in bright illumination. In mouse and human retina, rods greatly outnumber cones and consume more energy overall even in background light. In primates, however, the high density of cones in the fovea produces a pronounced peak of ATP utilization, which becomes particularly prominent in daylight and may make this part of the retina especially sensitive to changes in energy availability.
Assuntos
Retina/metabolismo , Células Fotorreceptoras Retinianas Cones/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Cálcio/metabolismo , GMP Cíclico/metabolismo , Canais de Cátion Regulados por Nucleotídeos Cíclicos/metabolismo , Fóvea Central/citologia , Fóvea Central/metabolismo , Humanos , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/metabolismo , Ativação do Canal Iônico , Luz , Camundongos , Terminações Pré-Sinápticas/metabolismo , Retina/citologia , Células Fotorreceptoras Retinianas Bastonetes/metabolismo , Sódio/metabolismoRESUMO
Ellis et al. show that retinal ON and OFF bipolar cells, and the novel metabotropic glutamate receptors of ON bipolar-cell dendrites, are both present in lamprey. They conclude that the fundamental organizing principle of separate ON and OFF pathways first appeared in the vertebrate visual system over 500 million years ago in the late Cambrian.
Assuntos
Evolução Biológica , Lampreias/fisiologia , Células Fotorreceptoras de Vertebrados/fisiologia , Retina/fisiologia , Animais , Técnicas de Patch-ClampRESUMO
KEY POINTS: Most vertebrate eyes have rods for dim-light vision and cones for brighter light and higher temporal sensitivity. Rods evolved from cone-like precursors through expression of different transduction genes or the same genes at different expression levels, but we do not know which molecular differences were most important. We approached this problem by analysing rod and cone responses with the same model but with different values for model parameters. We showed that, in addition to outer-segment volume, the most important differences between rods and cones are: (1) decreased transduction gain, reflecting smaller amplification in the G-protein cascade; (2) a faster rate of turnover of the second messenger cGMP in darkness; and (3) an accelerated rate of decay of the effector enzyme phosphodiesterase and perhaps also of activated visual pigment. We believe our analysis has identified the principal alterations during evolution responsible for the duplex retina. ABSTRACT: Most vertebrates have rod and cone photoreceptors, which differ in their sensitivity and response kinetics. We know that rods evolved from cone-like precursors through the expression of different transduction genes or the same genes at different levels, but we do not know which molecular differences were most important. We have approached this problem in mouse retina by analysing the kinetic differences between rod flash responses and recent voltage-clamp recordings of cone flash responses, using a model incorporating the principal features of photoreceptor transduction. We apply a novel method of analysis using the log-transform of the current, and we ask which of the model's dynamic parameters need be changed to transform the flash response of a rod into that of a cone. The most important changes are a decrease in the gain of the response, reflecting a reduction in amplification of the transduction cascade; an increase in the rate of turnover of cGMP in darkness; and an increase in the rate of decay of activated phosphodiesterase, with perhaps also an increase in the rate of decay of light-activated visual pigment. Although we cannot exclude other differences, and in particular alterations in the Ca2+ economy of the photoreceptors, we believe that we have identified the kinetic parameters principally responsible for the differences in the flash responses of the two kinds of photoreceptors, which were likely during evolution to have resulted in the duplex retina.
Assuntos
Células Fotorreceptoras Retinianas Cones , Células Fotorreceptoras Retinianas Bastonetes , Animais , Cinética , Camundongos , Retina , Pigmentos da RetinaRESUMO
Purpose: Cone photoreceptor function loss 3 (Gnat2cpfl3/cpfl3 or cpfl3) is a mouse model commonly used as a functional cones null from a naturally occurring mutation in the α-subunit of cone transducin (Gnat2). We nevertheless detected robust cone-mediated light responses from cpfl3 animals, which we now explore. Methods: Recordings were made from whole retina and from identified cones with whole-cell patch clamp in retinal slices. Relative levels of GNAT2 protein and numbers of cones in isolated retinas were compared between cpfl3, rod transducin knockout (Gnat1-/-), cpfl3/Gnat1-/- double mutants, and control C57Bl/6J age-matched mice at 4, 9, and 14 weeks of age. Results: Cones from cpfl3 and cpfl3/Gnat1-/- mice 2 to 3 months of age displayed normal dark currents but greatly reduced sensitivity and amplification constants. Responses decayed more slowly than in control (C57Bl/6J) mice, indicating an altered mechanism of inactivation. At dim light intensities rod responses could be recorded from cpfl3 cones, indicating intact rod/cone gap junctions. The cpfl3 and cpfl3/Gnat1-/- mice express two-fold less GNAT2 protein compared with C57 at 4 weeks, and a four-fold decrease by 14 weeks. This is accompanied by a small decrease in the number of cones. Conclusions: Cplf3 cones can respond to light with currents of normal amplitude and cannot be assumed to be a Gnat2 null. The decreased sensitivity and amplification rate of cones is not explained by a reduction in GNAT2 protein level, but instead by abnormal interactions of the mutant transducin with rhodopsin and the effector molecule, cGMP phosphodiesterase.
Assuntos
Regulação da Expressão Gênica , Proteínas Heterotriméricas de Ligação ao GTP/genética , Doenças Retinianas/genética , Transducina/genética , Visão Ocular/genética , Animais , Modelos Animais de Doenças , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mutação , Estimulação Luminosa , Células Fotorreceptoras Retinianas Cones/fisiologia , Doenças Retinianas/fisiopatologia , Células Fotorreceptoras Retinianas Bastonetes/fisiologia , Transdução de Sinais/genéticaRESUMO
Vertebrate photoreceptor cells respond to light through a closure of CNG channels located in the outer segment. Multiple voltage-sensitive channels in the photoreceptor inner segment serve to transform and transmit the light-induced outer-segment current response. Despite extensive studies in lower vertebrates, we do not know how these channels produce the photoresponse of mammalian photoreceptors. Here we examined these ionic conductances recorded from single mouse cones in unlabeled, dark-adapted retinal slices. First, we show measurements of the voltage dependence of the light response. After block of voltage-gated Ca2+ channels, the light-dependent current was nearly linear within the physiological range of voltages with constant chord conductance and a reversal potential similar to that previously determined in lower vertebrate photoreceptors. At a dark resting membrane potential of -45 mV, cones maintain a standing Ca2+ current (iCa) between 15 and 20 pA. We characterized the time and voltage dependence of iCa and a calcium-activated anion channel. After constitutive closure of the CNG channels by the nonhydrolysable analogue GTP-γ-S, we observed a light-dependent increase in iCa followed by a Ca2+-activated K+ current, both probably the result of feedback from horizontal cells. We also recorded the hyperpolarization-activated cyclic nucleotide-gated (HCN) conductance (ih) and measured its current-voltage relationship and reversal potential. With small hyperpolarizations, ih activated with a time constant of 25 ms; activation was speeded with larger hyperpolarizations. Finally, we characterized two voltage-gated K+-conductances (iK). Depolarizing steps beginning at -10 mV activated a transient, outwardly rectifying iK blocked by 4-AP and insensitive to TEA. A sustained iK isolated through subtraction was blocked by TEA but was insensitive to 4-AP. The sustained iK had a nearly linear voltage dependence throughout the physiological voltage range of the cone. Together these data constitute the first comprehensive study of the channel conductances of mouse photoreceptors.
Assuntos
Potenciais da Membrana/fisiologia , Células Fotorreceptoras Retinianas Cones/metabolismo , Animais , Cálcio/metabolismo , GMP Cíclico/metabolismo , Feminino , Ativação do Canal Iônico/fisiologia , Canais Iônicos/metabolismo , Masculino , Camundongos , Potássio/metabolismo , Retina/metabolismo , Células Fotorreceptoras Retinianas Bastonetes/metabolismo , Canais de Ânion Dependentes de Voltagem/metabolismoRESUMO
The lamprey is an important non-model vertebrate because it is an agnathan or jawless vertebrate and belongs to the superclass cyclostomata, a group that split off from the rest of the vertebrates 500 million years ago. Investigation of the lamprey retina may therefore reveal attributes of visual function that were characteristic of even the most primitive vertebrates. The rod and cone photoreceptors are a striking example, because the biochemistry and physiology of phototransduction is remarkably similar between lamprey and the rest of the vertebrates, including mammals. The fundamental mechanism of light sensation seems therefore to have emerged very early in the evolution of vertebrates in the late Cambrian. Some other characteristics of the retina are also similar and may be very old, but other features such as the morphology of ganglion cells are rather different in lamprey and other vertebrates. Even these differences may provide new insight into the various mechanisms vertebrates use for visual detection.
Assuntos
Lampreias/fisiologia , Retina/fisiologia , Células Fotorreceptoras Retinianas Cones/fisiologia , AnimaisRESUMO
We describe the first extensive study of voltage-clamp current responses of cone photoreceptors in unlabeled, dark-adapted mouse retina using only the position and appearance of cone somata as a guide. Identification was confirmed from morphology after dye filling. Photocurrents recorded from wild-type mouse cones were biphasic with a fast cone component and a slower rod component. The rod component could be eliminated with dim background light and was not present in mouse lines lacking the rod transducin-α subunit (Gnat1-/- ) or connexin 36 (Cx36-/- ). Cones from Gnat1-/- or Cx36-/- mice had resting membrane potentials between -45 and -55 mV, peak photocurrents of 20-25 picoamps (pA) at a membrane potential Vm = -50 mV, sensitivities 60-70 times smaller than rods, and a total membrane capacitance two to four times greater than rods. The rate of activation (amplification constant) was largely independent of the brightness of the flash and was 1-2 s-2, less than half that of rods. The role of Ca2+-dependent transduction modulation was investigated by recording from cones in mice lacking rod transducin (Gnat1), recoverin, and/or the guanylyl-cyclase-activating proteins (GCAPs). In confirmation of previous results, responses of Gnat1-/- ;Gcaps-/- cones and triple-mutant Gnat1-/- ;Gcaps-/- ;Rv-/- cones recovered more slowly both to light flashes and steps and were more sensitive than cones expressing the GCAPs. Cones from all four mouse lines showed significant recovery and escaped saturation even in bright background light. This recovery occurred too rapidly to be caused by pigment bleaching or metaII decay and appears to reflect some modulation of response inactivation in addition to those produced by recoverin and the GCAPs. Our experiments now make possible a more detailed understanding of the cellular physiology of mammalian cone photoreceptors and the role of conductances in the inner and outer segment in producing cone light responses.
Assuntos
Conexinas/metabolismo , Técnicas de Patch-Clamp/métodos , Células Fotorreceptoras Retinianas Cones/fisiologia , Transducina/metabolismo , Animais , Sinalização do Cálcio , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/metabolismo , Conexinas/genética , Camundongos , Camundongos Knockout , Mutação , Transducina/genética , Proteína delta-2 de Junções ComunicantesRESUMO
While rods in the mammalian retina regenerate rhodopsin through a well-characterized pathway in cells of the retinal pigment epithelium (RPE), cone visual pigments are thought to regenerate in part through an additional pathway in Müller cells of the neural retina. The proteins comprising this intrinsic retinal visual cycle are unknown. Here, we show that RGR opsin and retinol dehydrogenase-10 (Rdh10) convert all-trans-retinol to 11-cis-retinol during exposure to visible light. Isolated retinas from Rgr+/+ and Rgr-/- mice were exposed to continuous light, and cone photoresponses were recorded. Cones in Rgr-/- retinas lost sensitivity at a faster rate than cones in Rgr+/+ retinas. A similar effect was seen in Rgr+/+ retinas following treatment with the glial cell toxin, α-aminoadipic acid. These results show that RGR opsin is a critical component of the Müller cell visual cycle and that regeneration of cone visual pigment can be driven by light.
Assuntos
Células Ependimogliais/metabolismo , Proteínas do Olho/genética , Receptores Acoplados a Proteínas G/genética , Células Fotorreceptoras Retinianas Cones/metabolismo , Pigmentos da Retina/metabolismo , Ácido 2-Aminoadípico/farmacologia , Oxirredutases do Álcool/metabolismo , Oxirredutases do Álcool/efeitos da radiação , Animais , Células Ependimogliais/efeitos dos fármacos , Células Ependimogliais/efeitos da radiação , Antagonistas de Aminoácidos Excitatórios/farmacologia , Proteínas do Olho/metabolismo , Proteínas do Olho/efeitos da radiação , Luz , Camundongos , Camundongos Knockout , Receptores Acoplados a Proteínas G/metabolismo , Receptores Acoplados a Proteínas G/efeitos da radiação , Células Fotorreceptoras Retinianas Cones/efeitos da radiação , Pigmentos da Retina/efeitos da radiação , Vitamina A/metabolismoRESUMO
The retinal degeneration model rd10 contains a missense mutation of the catalytic PDE6 ß subunit, which hydrolyzes cGMP in response to light. This model produces cell death more slowly than others caused by PDE6 loss of function, making it of particular interest for studying potential therapeutics. We used morphology, biochemistry, and single-cell physiology to examine the mechanism of rd10 degeneration. Our results show that the mutation produces no alteration of Pde6b RNA but does dramatically decrease maximal and basal PDE6 activity, apparently caused by a decrease in protein stability and transport. The enzymatic properties of the remaining mutant PDE6 appear to be nearly normal. We demonstrate that an increase in free cGMP, which would result from decreased PDE6 activity and serve to increase opening of the cGMP-gated channels and calcium influx, is an underlying cause of cell death: degeneration of rd10/Cngb1-/- double mutants is slower than the parent rd10 line. Paradoxically, degeneration in rd10/Cngb1-/- is also slower than in Cngb1-/- This rescue is correlated with a lowering of cGMP content in Cngb1-/- retinas and suggests that it may be caused by mislocalization of active PDE6. Single-cell recordings from rd10 rods show that the rates of rise and decay of the response are significantly slower; simulations indicate that these changes are primarily the result of the decrease in PDE6 concentration and rod collecting area. Together, these results provide insights into the complex mechanisms that underlie rd10-mediated retinal degeneration and a cautionary note for analysis of therapeutic interventions.