2009 — 2010 |
Kefalov, Vladimir Jivkov |
R21Activity Code Description: To encourage the development of new research activities in categorical program areas. (Support generally is restricted in level of support and in time.) |
Calcium and Adaptation in Mammalian Cone Photoreceptors
DESCRIPTION (provided by applicant): Cone photoreceptors function under daylight conditions and are essential for color perception and vision with high temporal and spatial resolution. A remarkable feature of cones is their ability to remain functional in bright light which requires powerful adaptation mechanisms of their phototransduction cascade. In rods, light adaptation is mediated by calcium and the mechanisms by which reduction in calcium upon photo-activation modulates phototransduction are well understood. While photo-activation also triggers a decline in calcium in cones, the molecular mechanisms by which this exerts negative feedback on cone phototransduction are not known. The experiments described here will employ physiological studies from available genetically modified mice to investigate the mechanisms of modulation of the cone phototransduction cascade by calcium. Specifically, we will test the hypothesis that regulation of visual pigment phosphorylation by rhodopsin kinase via recoverin is a component of the calcium feedback in mouse cones and modulates their phototransduction in darkness and during light adaptation. We will also test the hypothesis that regulation of cGMP synthesis by guanylyl cyclase via Guanylyl Cyclase Activating Proteins (GCAPs) is a major component of the calcium feedback in mouse cones and modulates their phototransduction in darkness and during light adaptation. Together, these experiments will help us determine the mechanisms that allow cones to adapt to a very wide range of light intensities and remain functional throughout the day, even in bright light. PUBLIC HEALTH RELEVANCE: The experiments outlined in this proposal seek to establish the mechanisms that enable mammalian cones to function in bright light, an essential property for the photoreceptors that mediate daytime vision. These studies will help us understand the role of calcium in regulating mammalian cone function under normal and pathological conditions.
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0.958 |
2009 — 2017 |
Kefalov, Vladimir Jivkov |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Mechanisms of Adaptation in Mammalian Cones
DESCRIPTION (provided by applicant): Cone photoreceptors function under bright light conditions and are essential for color perception and vision with high temporal and spatial resolution. Remarkably, unlike rods, cones remain functional even in steady bright light and dark adapt rapidly. Both of these properties require rapid recycling of chromophore for regeneration of cone visual pigment. Biochemical studies and shortcomings of the canonical pigment epithelium pathway for chromophore recycling indicate the possible existence of a second, cone- specific chromophore pathway located in the retina and independent of the pigment epithelium. The function of such a pathway under physiological conditions, its role in photoreceptor physiology, and its regulation have not been investigated. We propose to use single-cell and whole retina recordings from mouse photoreceptors to characterize the physiological function of this novel visual cycle. Specifically, we will determine the ability of mammalian retina to promote pigment regeneration and dark adaptation in cones independently of the pigment epithelium. We will establish the role of the mammalian retina visual cycle in extending the dynamic range of cones during background adaptation and in accelerating the recovery of cone sensitivity during dark adaptation. We will determine whether the specificity of the mammalian retina visual cycle is based on the ability of cones, and not rods, to oxidize 11-cis retinol, recycled within the retina, into 11-cis retinal and use it for pigment regeneration. We will use available genetically modified mice and pharmacological tools to characterize key steps in the pathway and their modulation by chromophore-binding proteins expressed in the retina. Collectively, the experiments outlined in this proposal seek to establish the mechanisms that enable mammalian cones to function in rapidly varying light conditions, an essential property for the photoreceptors that mediate daytime vision. In addition to advancing the understanding of cone cell biology, our studies of the mammalian retina visual cycle have potential clinical implications. Mutations in the chromophore-binding proteins investigated in this study have been associated with multiple visual disorders including Stargardt disease, cone-rod dystrophy, and macular degeneration. No treatments currently exist for these disorders. Our experiments will lay the foundation for understanding how specific defects in the retina visual cycle produce cone-related retinal disorders, as well as for the development of new treatments targeting specifically the function of cones. PUBLIC HEALTH RELEVANCE: The experiments outlined in this proposal seek to establish the mechanisms that enable mammalian cones to function in bright light, an essential property for the photoreceptors that mediate daytime vision. These studies will help us understand the mechanisms that regulate mammalian cone function under normal and pathological conditions.
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0.958 |
2016 — 2019 |
Kefalov, Vladimir Jivkov |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Pigment Dephosphorylation in Mammalian Rod and Cone Photoreceptors
ABSTRACT The state of phosphorylation mediates the activity of many G protein-coupled receptors. In photoreceptors, the phosphorylation of the visual pigment has been well characterized. However, an often-overlooked step in the resetting of the visual pigment following its photoactivation is its dephosphorylation. Despite remarkable progress in understanding G protein signaling in general, and phototransduction in particular, the enzyme responsible for catalyzing this reaction in vivo remains unknown. Also unknown is the role of visual pigment dephosphorylation in modulating the kinetics of dark adaptation or the susceptibility of photoreceptors to light damage. We have generated mice with loxP-flanked catalytic ?-subunit of PP2A (Ppp2cafl/fl) which will allow us to investigate the role of this enzyme in pigment dephosphorylation and the function and survival of mammalian photoreceptors. Crossing these animals with mice expressing Cre recombinase selectively in rod or cone photoreceptors has allowed us to generate rod- and cone-specific PP2A C? knockout mice. We will perform experiments to determine whether pigment dephosphorylation is suppressed in these mice. These experiments will establish whether PP2A is the elusive pigment phosphatase in mammalian rods and cones. We will also determine how the deletion of PP2A C? affects the expression profile, morphology, and survival of photoreceptors. We will also analyze the light responses of PP2A C?-deficient mouse rods and cones to determine the role of PP2A in modulating phototransduction. By in vivo ERG recordings, we will also determine whether the deletion of PP2A affects the kinetics of photoreceptor pigment regeneration and dark adaptation. These experiments will establish the role of pigment dephosphorylation in regulating the dark adaptation of mammalian rods and cones. The link between pigment phosphorylation and photoreceptor degeneration will also be explored using a combination on mutant mice lacking arrestin, rhodopsin kinase, and PP2A. Finally, we will also test the hypothesis that light-induced phosphorylation of ground state cone visual pigment reduces the overall light sensitivity and represents a novel mechanism for mammalian cone background light adaptation. Collectively, our experiments will establish the molecular mechanism for visual pigment dephosphorylation in mammalian photoreceptors. They will also determine the role of pigment dephosphorylation in controlling the kinetics of mammalian rod and cone dark adaptation, as well as the therapeutic potential of modulating this enzymatic reaction in light- or opsin-induced retinal degeneration.
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0.958 |
2020 |
Kefalov, Vladimir Jivkov Palczewski, Krzysztof (co-PI) [⬀] |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Opsin Signaling in Mammalian Rod Photoreceptors
ABSTRACT Light detection in rod photoreceptors is mediated by a G protein-signaling cascade triggered when the visual pigment rhodopsin, a prototypical G protein-coupled receptor, is activated by light and initiates the rod light response. Eventually, the photoactivated rhodopsin decays to all-trans-retinal and free opsin. The chromophore-free opsin produces persistent transduction activity even in darkness. This, in turn desensitizes the photoreceptors and limits our ability to see following exposure to bright light. Abnormally high opsin activity due to opsin mutations or slow pigment regeneration can be detrimental to rod function and survival. Despite the role of opsin activity in modulating rod physiology in normal and disease conditions, the molecular mechanism by which opsin stimulates rod transduction has remained unclear. The prevailing view is that each opsin has low uniform constitutive activity. We will evaluate an alternative hypothesis that, similar to rhodopsin, opsin exists in equilibrium between a distinct inactive state and a rare but highly efficient active state. This hypothesis is based on our preliminary data showing that introduction of a small amount of free opsin by rhodopsin bleaching results in the generation of discrete, photoresponse-like events in mouse rods. We will perform experiments to determine the amplitude and kinetics of the quantal response produced by a single opsin molecule in mouse, primate, and human rod photoreceptors. We will also determine the mechanism of modulation of opsin activity by non-covalent binding of chromophore analogs and chaperones, and whether blocking opsin signaling either by quenching with chaperones, or by using 5- or 6-locked rhodopsin that does not dissociate upon photoactivation restores the sensitivity of chromophore-deficient rods. Finally, we will perform experiments to determine the role of phosphorylation and arrestin binding in the inactivation of opsin signaling. These experiments will establish the molecular mechanisms by which opsin activates the rod transduction cascade to produce bleaching adaptation. They will also help us understand how this activity can be modulated pharmacologically, potentially leading to the development of treatments for a range of opsin-related visual disorders such as congenital stationary night blindness and retinitis pigmentosa.
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0.958 |
2020 — 2021 |
Kefalov, Vladimir Jivkov |
P30Activity Code Description: To support shared resources and facilities for categorical research by a number of investigators from different disciplines who provide a multidisciplinary approach to a joint research effort or from the same discipline who focus on a common research problem. The core grant is integrated with the center's component projects or program projects, though funded independently from them. This support, by providing more accessible resources, is expected to assure a greater productivity than from the separate projects and program projects. |
Visual Function Testing Core
Project Summary A Visual Function Testing Core will provide expertise, instrumentation, and training on technologies used to quantify visual performance in mice and other model systems. ERG, VEP and optometry analysis are supported. This core also includes technical support for the fabrication, diagnosis and repair of electronic equipment. Provision of these support services and resources will greatly enhance the research capabilities of investigators at Washington University and will facilitate collaboration among new and established vision scientists.
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0.958 |