Rhodopsin-mediated phospholipid flipping

描述(由申请者提供)：视网膜中的脂类运输对视力至关重要。类维甲酸必须在感光细胞和视网膜色素上皮细胞之间快速移动，才能在光线照射到视网膜后再生视觉色素视紫红质。这种依赖于运输的再生过程被称为视觉周期，对于持续的视觉是必不可少的。脂质转运的缺陷会导致视网膜病变；例如，Stargardt黄斑营养不良是由于无法将视黄醇-磷脂加合物转移到光感受器盘上而引起的。我们最近在视网膜内的脂质运输中发现了一个令人惊讶的新参与者：我们的生化重建研究表明，视紫红质是一种不依赖于ATP的磷脂转运体(Flippase)，能够在膜双层上快速移动磷脂。这一发现为以前在盘膜中观察到的磷脂触发器提供了神秘的分子基础，并赋予了视紫红质除了众所周知的光转导功能之外的新活性。我们在此应用程序中的目标 是为了破译视紫红质通过膜双层翻转脂质的分子机制。我们建议确定视紫红质跨膜螺旋束的结构和动力学特征，这些特征是其翻转酶活性所必需的，并确定它是否受其膜环境的调节，特别是具有多不饱和酰链的胆固醇和磷脂。我们认为，视紫红质的脂翻转酶活性对DIC膜的动态平衡至关重要，因为它纠正了由ATP驱动的脂类转运蛋白引起的磷脂失衡，包括Stargardt病转运蛋白ABCA4，该转运蛋白将磷脂从管腔泵入椎间盘的细胞质表面。我们提出的阐明视紫红质翻转机制的建议具有非常重要的意义，因为它不仅将建立一个新的膜转运机制，而且也是理解视网膜中脂类稳态的关键，这与视网膜变性有关。视紫红质突变与视网膜色素变性有关，但许多视紫红质突变的潜在致病机制尚不清楚。我们拟议的研究有可能揭示一些不明原因的突变影响Flippase活性，从而澄清这种视网膜疾病几十年来一直未解决的问题。最后，视紫红质是一种典型的G蛋白偶联受体。由于其他GPCR已被证明具有磷脂翻转酶活性，我们在这里的发现将产生超出视觉系统的影响。

DESCRIPTION (provided by applicant): Lipid trafficking in the retina is crucial for vision. Retinoids must move rapidly between photoreceptor cells and retinal pigment epithelial cells to regenerate the visual pigment rhodopsin after light impinges on the retina. This trafficking-dependent regeneration process is termed the visual cycle and is essential for continuous vision. Defects in lipid trafficking result in retinopathies; for example, Stargardt's macular dystrophy is caused by the inability to translocate a retinoid-phospholipid adduct across photoreceptor discs. We recently discovered a surprising new player in lipid transport within the retina: our biochemical reconstitution studies revealed that rhodopsin is an ATP-independent phospholipid translocator (flippase) capable of moving phospholipids rapidly across a membrane bilayer. This discovery provides the molecular basis for previous enigmatic observations of phospholipid flip-flop in disc membranes, and assigns a novel activity to rhodopsin in addition to its well-known function in phototransduction. Our goal in this application is to decipher the molecular mechanism by which rhodopsin flips lipids across a membrane bilayer. We propose to identify structural and dynamic features of rhodopsin's transmembrane helical bundle that are necessary for its flippase activity and also determine whether it is regulated by its membrane environment, specifically cholesterol and phospholipids with polyunsaturated acyl chains. We believe that rhodopsin's lipid flippase activity is critical for dic membrane homeostasis as it corrects the phospholipid imbalance caused by ATP-driven lipid transporters, including the Stargardt's disease transporter' ABCA4, that pump phospholipids from the lumen to the cytoplasmic face of discs. Our proposal to elucidate rhodopsin's flipping mechanism is highly significant because it will not only establish a new mechanistic paradigm in membrane transport but is also key to understanding lipid homeostasis in the retina, with implications for retinal degeneration. Mutations in rhodopsin are linked to retinitis pigmentosa, but the underlying disease-causing mechanism for many of the rhodopsin mutations is not known. Our proposed studies have the potential to reveal that some of the unexplained mutations affect flippase activity, thus clarifying aspects of this retinal disease that have remained unresolved for decades. Finally, rhodopsin is a prototypical G protein-coupled receptor (GPCR). As other GPCRs have been shown to have phospholipid flippase activity, our discoveries here will have implications beyond the visual system.