Opsin signaling in mammalian rod photoreceptors

摘要 视杆细胞光感受器中的光检测是由G蛋白信号级联介导的， 视色素视紫红质是一种典型的G蛋白偶联受体，它被光激活， 视杆细胞的光反应最终，光活化的视紫红质衰变为全反式视网膜和游离的 视蛋白无发色团的视蛋白即使在黑暗中也产生持续的转导活性。而这 转光使光感受器脱敏，并限制了我们在暴露于强光下后的视觉能力。 由于视蛋白突变或色素再生缓慢而导致的异常高的视蛋白活性可能是有害的 视杆细胞的功能和存活尽管视蛋白活性在调节正常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，每种视蛋白具有低的均匀组成活性。我们将 评估一个替代假设，类似于视紫红质，视蛋白存在于平衡之间， 独特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 初步数据显示通过视紫红质漂白引入少量游离视蛋白 导致在小鼠视杆细胞中产生离散的光响应样事件。我们将执行 实验，以确定由单一的量子响应产生的振幅和动力学， 小鼠、灵长类动物和人类视杆细胞感光器中的视蛋白分子。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， 分子伴侣，以及是否通过用分子伴侣猝灭或通过使用5- 在光活化时不解离的或6-锁定的视紫红质恢复了对 缺乏发色团的杆。最后，我们将进行实验以确定 磷酸化和抑制蛋白结合在视蛋白信号传导失活中的作用。这些实验将 建立视蛋白激活视杆细胞转导级联产生 漂白适应它们还将帮助我们了解如何调节这种活动 这可能导致治疗一系列视蛋白相关疾病的发展。 视觉障碍，如先天性静止性夜盲症和视网膜色素变性。

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.