LIPID MODULATION OF RHODOPSIN SIGNALING IN MEMBRANES

描述（由申请人提供）：在这里，我们将测试视网膜杆盘膜脂质成分通过其对涉及视紫红质的信号传导和放大过程的影响来控制视觉功能的假设。重点将放在膜环境中的作用，在调制的Meta I-Meta II平衡，这是在视觉兴奋的信号事件。视网膜视杆细胞膜中含有非常丰富的磷脂，其中含有高度多不饱和脂肪酸，包括二十二碳六烯酸（DHA; 22：6-omega-3）和花生四烯酸（20：4-omega-6）。发现视觉功能的改变发生在必需脂肪酸缺乏症中。生物物理方法将表征膜脂质对视紫红质的Meta I}Meta II转变的影响。具体目标是应用多学科方法（1）鉴定作为视紫红质信号传导的激动剂或拮抗剂的膜脂质;（2）阐明影响视紫红质光化学功能的膜脂质双层的性质;（3）阐明脂质多不饱和在视紫红质活化中的作用;（4）发现膜的静电性质如何支配视紫红质活化;（5）阐明视紫红质的光化学功能。以及（5）确定膜脂对视紫红质的影响如何在视觉信号传导中被放大。基于光学多通道分析仪（OMA）的时间分辨多波长方法将用于研究视紫红质激活的动力学和机制。此外，傅里叶变换红外（FTIR），荧光共振能量转移（FRET），和等离子体波导共振（PWR）光谱将阐明视网膜环境，蛋白质构象，和低聚或关联的视紫红质在黑暗中，Meta I，和Meta II状态。一个新的柔性表面模型（FSM）将提供一个框架，了解视紫红质的信号功能是如何驱动的膜磷脂的非特异性，包括膜脂质曲率和疏水力内的双层。FSM描述的曲率变形能量的平衡方面的脂质-蛋白质的相互作用，由于弹性应力/应变的双层，与蛋白质脂质界面的溶剂化能。另一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性质的双电层。将研究多不饱和膜磷脂对视觉光反应后期放大阶段的影响，包括G蛋白（转导素）与光解视紫红质的结合和激活，以及随后的cGMP磷酸二酯酶激活。以这种方式，一个真正全面的图片的触发和放大步骤的视觉过程将提供在膜水平上的饮食调查的基本？3脂肪酸缺乏症。公共卫生相关性：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，包括哺乳动物神经系统的独特模型。目前的知识表明，长链多不饱和脂肪酸来源于必需的？3脂肪酸在涉及人类婴儿的视网膜和大脑发育中起重要作用。此外，多不饱和脂质参与疾病，如帕金森病、心血管疾病、癌症、衰老和其他生理和病理异常。所提出的膜脂双层对视紫红质活性影响的体外研究将测试用于解释在膜水平上视觉系统中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质在人类中枢神经系统功能和功能障碍中的作用有关，并伴随着可能具有最终治疗益处的见解。

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.