Opsin signaling in mammalian rod photoreceptors

摘要 杆状感光器中的光检测是由G蛋白信号级联介导的，当 视紫红质，一种典型的G蛋白偶联受体，被光激活并启动 杆状光的反应。最终，光激活的视紫红质会分解成全视网膜和游离的 视黄素。无生色团的视蛋白即使在黑暗中也能产生持续的转导活性。这个，在 TURN会使感光器变得不敏感，并限制我们在暴露在强光下后的视力。 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。 以提高功能和生存能力。尽管视蛋白活性在调节视杆细胞生理中的作用 和疾病条件下，视蛋白刺激视杆转导的分子机制有 目前仍不清楚。流行的观点是，每种视蛋白都具有较低的统一构成活性。我们会 评估另一种假设，即与视紫红质类似，视黄素存在于 独特的非活动状态和罕见但高效的活动状态。这一假设是基于我们的 初步数据显示通过视紫红质漂白引入少量游离视黄素 导致在鼠标棒中产生离散的、类似光反应的事件。我们将表演 确定单个分子产生的量子响应的幅度和动力学的实验 小鼠、灵长类和人类杆状感光器中的视蛋白分子。我们还将确定 发色团及其类似物非共价结合调节视蛋白活性的机制 以及是否通过伴侣猝灭或使用5-羟色胺来阻断视蛋白信号传导。 或6-锁定视紫红质，在光激活时不解离，恢复对 生色团缺乏棒。最后，我们将进行实验，以确定 视蛋白信号失活中的磷酸化和arrestin结合。这些实验将 建立视蛋白激活杆状转导级联反应的分子机制 漂白适应。它们还将帮助我们理解这种活动是如何被调节的 从药理上讲，可能导致开发一系列视蛋白相关的治疗方法 视力障碍，如先天性静止性夜盲和视网膜色素变性。

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.