Opsin signaling in mammalian rod photoreceptors

哺乳动物视杆光感受器中的视蛋白信号传导

• 批准号: 10475531
• 负责人: Vladimir Jivkov Kefalov
• 金额: $ 38.07万
• 依托单位: UNIVERSITY OF CALIFORNIA-IRVINE
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-06-01 至 2024-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10475531
• 关键词: 11 cis Retinal; Affect; Arrestins; Belief; Binding; Darkness; Data; Detection; Disease; Equilibrium; Event; Exposure to; G-Protein-Coupled Receptors; GRK1 gene; GTP-Binding Protein alpha Subunits, Gs; Generations; Human; Individual; Kinetics; Light; Measures; Mediating; Molecular; Molecular Chaperones; Mus; Mutation; Natural regeneration; Night Blindness; Opsin; Pharmacological Treatment; Pharmacology; Phosphorylation; Photobleaching; Photoreceptors; Physiology; Pigments; Primates; Process; Recycling; Research; Retina; Retinal Pigments; Retinitis Pigmentosa; Rhodopsin; Rod; Role; Shapes; Signal Transduction; Signaling Protein; Testing; Time; Vision Disorders; analog; base; chromophore; desensitization; experimental study; photoactivation; quantum; response; retinal rods; therapy development; transduction efficiency

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.

抽象的 杆状光感受器中的光检测是由 G 蛋白信号级联介导的，当 视色素视紫红质是一种典型的 G 蛋白偶联受体，被光激活并启动 棒光响应。最终，光激活的视紫红质衰变为全反式视网膜并游离 视蛋白。即使在黑暗中，无发色团的视蛋白也能产生持续的转导活性。这，在 转变会使光感受器变得不敏感，并限制我们暴露在强光下的视力。 由于视蛋白突变或色素再生缓慢导致的视蛋白活性异常高可能是有害的 杆功能和生存。尽管视蛋白活性在正常情况下调节视杆细胞生理学中的作用 和疾病条件下，视蛋白刺激视杆细胞转导的分子机制 仍不清楚。普遍的观点是每种视蛋白具有低均匀的组成活性。我们将 评估另一种假设，即与视紫红质类似，视蛋白存在于 明显的非活动状态和罕见但高效的活动状态。这个假设是基于我们的 初步数据表明，通过视紫红质漂白引入少量游离视蛋白 导致小鼠视杆细胞中产生离散的、类似光响应的事件。我们将表演 确定单个粒子产生的量子响应的幅度和动力学的实验 小鼠、灵长类动物和人类视杆光感受器中的视蛋白分子。我们还将确定 通过发色团类似物的非共价结合调节视蛋白活性的机制和 分子伴侣，以及是否通过用分子伴侣淬灭或使用 5- 来阻断视蛋白信号传导 或 6 锁视紫红质在光激活时不会解离，可恢复 发色团缺陷棒。最后，我们将进行实验来确定 视蛋白信号传导失活中的磷酸化和抑制蛋白结合。这些实验将 建立视蛋白激活视杆转导级联产生的分子机制 漂白适应。它们还将帮助我们了解如何调节此活动 药理学上，可能导致一系列视蛋白相关治疗方法的开发 视力障碍，例如先天性静止性夜盲症和色素性视网膜炎。