LIPID MODULATION OF RHODOPSIN SIGNALING IN MEMBRANES

描述(申请人提供)：在这里，我们将测试视网膜视盘膜脂成分通过它们对涉及视紫红质的信号和放大过程的影响来控制视觉功能的假设。重点将放在膜环境在调节Meta I-Meta II平衡中的作用，Meta I-Meta II平衡是视觉兴奋中的信号事件。视网膜视杆盘膜富含含有高度多不饱和脂肪酸的磷脂，包括二十二碳六烯酸(DHA；22：6-omega-3)和花生四烯酸(20：4-omega-6)。视功能的改变被发现发生在必需脂肪酸缺乏症中。生物物理方法将表征膜脂对视紫红质的Meta I和Meta II转变的影响。其具体目标是应用多学科方法来：(1)确定起视紫红质信号激动剂或拮抗剂作用的膜脂；(2)阐明影响视紫红质光化学功能的膜脂双分子层的性质；(3)阐明脂质多不饱和在视紫红质激活中的作用；(4)发现膜的静电性质如何控制视紫红质的激活；以及(5)建立膜脂对视紫红质的影响如何在视觉信号中被放大。基于光学多通道分析仪(OMA)的时间分辨多波长方法将被用来研究视紫红质激活的动力学和机制。此外，傅立叶变换红外光谱(FTIR)、荧光共振能量转移(FRET)和等离子体波导管共振(PWR)光谱将阐明视网膜环境、蛋白质构象以及视紫红质在暗状态、Meta I状态和Meta II状态下的寡聚或结合。一个新的柔性表面模型(FSM)将提供一个框架，用于理解视紫红质的信号功能是如何由膜磷脂的非特异性属性驱动的，包括膜脂曲率和双层内的疏水性作用力。有限状态机通过双层的弹性应力/应变引起的曲率变形能与蛋白质脂界面的溶剂化能之间的平衡来描述脂-蛋白质相互作用。另一个方面需要双层静电的相互作用，包括表面电荷密度和具有上述双层属性的双电层。多不饱和膜磷脂对视觉光反应后期放大阶段的影响将被研究，包括G蛋白(转导蛋白)与光解视紫红质的结合和激活，以及随后cGMP磷酸二酯酶的激活。通过这种方式，将在膜水平上提供与人类必需脂肪酸缺乏症的饮食调查有关的视觉过程的触发和放大步骤的真正全面的图景。与公共健康相关：拟议的研究将调查视网膜中必需脂肪酸缺乏的分子基础，视网膜是大脑的一部分，由哺乳动物神经系统唯一可访问的模型组成。目前的知识表明，源于必需脂肪酸的长链多不饱和脂肪酸在涉及人类婴儿的视网膜和大脑发育中发挥着重要作用。此外，多不饱和脂肪还与帕金森氏症、心血管疾病、癌症、衰老和其他生理和病理异常有关。拟议的膜脂双层对视紫红质活性影响的体外研究将测试在膜水平上解释视觉系统中必需脂肪酸(必需脂肪酸)缺乏的影响的特定框架。这项工作与多不饱和脂肪在人类中枢神经系统功能和功能障碍中的作用有关，并伴随着可能最终对治疗有益的见解。

DESCRIPTION (provided by applicant): Here we shall test the hypothesis that the retinal rod disk membrane lipid constituents govern visual function through their influences on signaling and amplification processes involving rhodopsin. Emphasis will be placed on the role of the membrane environment in modulating the Meta I-Meta II equilibrium, which is the signaling event in visual excitation. The retinal rod disk membranes are extraordinarily abundant in phospholipids containing highly polyunsaturated fatty acids, including docosahexaenoic acid (DHA; 22:6-omega-3) and arachidonic acid (20:4-omega-6). Alterations of visual function are found to occur in essential fatty acid deficiency. Biophysical methods will characterize the influences of membrane lipids on the Meta I}Meta II transition of rhodopsin. Specific Aims are to apply a multidisciplinary approach to (1) identify the membrane lipids that function as agonists or antagonists of rhodopsin signaling; (2) elucidate the properties of membrane lipid bilayers that influence the photochemical function of rhodopsin; (3) illuminate the role of lipid polyunsaturation in rhodopsin activation; (4) discover how electrostatic properties of the membrane govern rhodopsin activation; and (5) establish how the membrane lipid influences on rhodopsin are amplified in visual signaling. A time-resolved multi-wavelength approach based on an optical multi-channel analyzer (OMA) will be used to study the kinetics and mechanism of rhodopsin activation. In addition, Fourier transform infrared (FTIR), fluorescence resonance energy transfer (FRET), and plasmon waveguide resonance (PWR) spectroscopy will elucidate the retinal environment, protein conformation, and oligomerization or association of rhodopsin in the dark, Meta I, and Meta II states. A new flexible surface model (FSM) will provide a framework for understanding how the signaling function of rhodopsin is driven by non-specific properties of the membrane phospholipids, including membrane lipid curvature and hydrophobic forces within the bilayer. The FSM describes the lipid-protein interactions in terms of a balance of the curvature deformation energy, due to elastic stress/strain of the bilayer, with the solvation energy of the proteolipid interface. An additional aspect entails the interplay of the bilayer electrostatics including the surface charge density and the electrical double layer with the above bilayer properties. The influences of polyunsaturated membrane phospholipids on later amplification stages of the visual photoresponse will be investigated, including the binding and activation of the G protein (transducin) to photolyzed rhodopsin, and subsequent activation of cGMP phosphodiesterase. In this manner, a truly comprehensive picture of the triggering and amplification steps of the visual process will be provided at the membrane level in relation to dietary investigations of essential ?3 fatty acid deficiency in humans. PUBLIC HEALTH RELEVANCE: The proposed research will investigate the molecular basis for essential fatty acid deficiency in the retina, which is part of the brain and comprises a uniquely accessible model for the mammalian nervous system. Current knowledge indicates that long chain polyunsaturated fatty acids derived from essential ?3 fatty acids play an important role in retinal and brain development involving human infants. Moreover, polyunsaturated lipids are involved in diseases such as Parkinson's disease, cardiovascular disease, cancer, aging, and other physiological and pathological anomalies. The proposed in vitro studies of the influence of the membrane lipid bilayer on rhodopsin activity will test a specific framework for explaining the effects of essential fatty acid) deficiency in the visual system at the membrane level. This work is pertinent to the role of polyunsaturated lipids in the function and dysfunction of central nervous system of humans with attendant insights that may be of eventual therapeutic benefit.