Physiology of Photoreceptors

描述（由申请人提供）：本提案的目的是了解哺乳动物感光器适应背景光和漂白的生理机制，以及在眼部疾病（如维生素A缺乏、某些形式的莱伯斯黑蒙和色素性视网膜炎）中产生感光器变性的过程。我们将使用抽吸电极记录和激光点显微镜技术，在与分子生物学家合作的情况下，在来自小鼠的单个分离的视杆上使用荧光指示剂染料，这些视杆已被遗传工程改造为缺乏某些转导蛋白，如鸟苷酸环化酶激活蛋白（GCAP）或RPE 65视网膜异构酶，或含有具有定点突变的转导蛋白，如磷酸二酯酶γ T35 A。在一些实验中，记录将从其中转导级联的关键蛋白质过度或不足表达的视杆中进行。我们的第一个具体目标直接解决了国家眼科研究所的计划目标之一，即“使用分子和生理方法来研究光感受器的光适应”。我们将试图发现和划定机制，负责在视杆反应波形和敏感性的变化，在背景光，包括GCAP依赖的Ca 2 +/环化酶反馈，调制的TaGTP水解速率，并通过recoverin调节视紫红质寿命。我们的第二个具体目标涉及NEI的另一个项目目标，即研究光感受器的适应性，“特别强调视觉周期”。我们将首次定量测量哺乳动物视杆细胞对色素漂白的适应性，并测试在生理条件下漂白光释放的全反式视网膜阻断视杆细胞外段通道的可能性。我们的第三个具体目标涉及NEI的另一个项目目标，即“确定遗传性和视网膜退行性疾病的病理生理机制”。我们将探讨持续激活过程中视网膜变性的可能机制，如维生素A缺乏和某些形式的莱伯斯黑蒙和视网膜色素变性，包括氧毒性和长期暴露于低游离钙。我们将试图确定的作用，钙在触发细胞凋亡的光感受器在杆中，无论是环核苷酸门控通道或Na+/Ca 2 +-K+转运蛋白已被过度表达或表达不足，以确定外段Ca 2+浓度和感光细胞变性率之间的关系。公共卫生相关性：绝大多数视网膜疾病是由光感受器的紊乱或变性引起的，光感受器是眼睛中将光转换成电信号的细胞。该提案旨在了解感光功能的基本机制，以解释为什么遗传性夜盲症和某些形式的遗传性视网膜变性的视力异常。只有了解为什么光感受器在这些疾病中表现异常，我们才有希望了解如何治愈它们并恢复正常视力。

DESCRIPTION (provided by applicant): The goal of this proposal is to understand the physiological mechanisms responsible for adaptation to background light and to bleaches in a mammalian photoreceptor, and the processes that produce photoreceptor degeneration in ocular diseases such as vitamin A deprivation, certain forms of Lebers amaurosis, and retinitis pigmentosa. We will use the techniques of suction-electrode recording and laser spot microscopy with fluorescent indicator dyes on single, isolated rods from mice which, in collaboration with molecular biologists, have been genetically engineered either to lack certain transduction proteins such as the guanylyl cyclase activating proteins (GCAPs) or the RPE65 retinal isomerase, or to contain transduction proteins with site-directed mutations, such as phosphodiesterase gamma T35A. In some experiments recordings will be made from rods in which critical proteins of the transduction cascade are either over or under expressed. Our first specific aim addresses directly one of the program goals of the National Eye Institute to "use both molecular and physiological approaches to study light adaptation in photoreceptors". We shall attempt to discover and delimit mechanisms responsible for changes in rod response waveform and sensitivity in background light, including GCAP-dependent Ca2+/cyclase feedback, modulation of TaGTP hydrolysis rate, and regulation of rhodopsin lifetime via recoverin. Our second specific aim addresses another of the program goals of the NEI to study adaptation in photoreceptors "with particular emphasis on the visual cycle". We will make the first quantitative measurements of adaptation of mammalian rods to pigment bleaching and test the possibility that all-trans retinal released by bleaching light blocks channels of the rod outer segment under physiological conditions. Our third specific aim addresses another of the program goals of the NEI to "determine the pathophysiological mechanisms underlying inherited and retinal degenerative diseases". We will explore possible mechanisms of retinal degeneration during continuous activation, as occurs in vitamin A deprivation and some forms of Lebers amaurosis and retinitis pigmentosa, including oxygen toxicity and prolonged exposure to low free-Ca2+. We shall attempt to define the role of Ca2+ in triggering apoptosis in photoreceptors in rods in which either the cyclic nucleotide-gated channels or the Na+/Ca2+-K+ transporters have been over-expressed or under-expressed, in order to define the relationship between outer segment Ca2+ concentration and the rate of photoreceptor degeneration. PUBLIC HEALTH RELEVANCE: The great majority of diseases of the retina are caused by disorder or degeneration of the photoreceptors, the cells in the eye which convert light into an electrical signal. This proposal seeks to understand basic mechanisms of photoreceptor function, with a view to explaining why vision is abnormal in hereditary night blindness and certain forms of inherited retinal degeneration. Only by understanding why the photoreceptors behave abnormally in these diseases can we hope to learn how to cure them and restore normal vision.