Rhodopsin-mediated phospholipid flipping

描述（由申请人提供）：视网膜中的脂质运输对视力至关重要。类维生素A必须在感光细胞和视网膜色素上皮细胞之间快速移动，以在光照射到视网膜上后再生视色素视紫红质。这种依赖于交通的再生过程被称为视觉周期，对连续视觉至关重要。脂质运输的缺陷导致视网膜病变;例如，Stargardt黄斑营养不良是由不能将类维生素A-磷脂加合物跨感光盘移位引起的。我们最近发现了视网膜内脂质转运的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ATP非依赖性磷脂转运蛋白（翻转酶），能够快速移动磷脂穿过膜双层。这一发现提供了分子基础，以前神秘的观察磷脂翻转光盘膜，并指定一个新的活动视紫红质除了其众所周知的功能，在光转导。我们在此应用程序中的目标 是破译视紫红质将脂质翻转穿过膜双层的分子机制。我们建议确定视紫红质的跨膜螺旋束的结构和动力学特征，这是必要的翻转酶活性，并确定它是否是由其膜环境，特别是胆固醇和磷脂与多不饱和酰基链的调节。我们相信视紫红质的脂质翻转酶活性对于dic膜稳态是至关重要的，因为它纠正了由ATP驱动的脂质转运蛋白引起的磷脂不平衡，包括Stargardt病转运蛋白ABCA 4，其将磷脂从管腔泵送到椎间盘的细胞质面。我们的建议，以阐明视紫红质的翻转机制是非常重要的，因为它不仅将建立一个新的机制范式膜运输，但也是关键，了解脂质稳态在视网膜，视网膜变性的影响。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。我们提出的研究有可能揭示一些无法解释的突变会影响翻转酶的活性，从而澄清这种视网膜疾病几十年来一直没有解决的问题。最后，视紫红质是一种原型G蛋白偶联受体（GPCR）。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对视觉系统以外的领域产生影响。

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.