Studies of Erythrocyte Membrane Structure

描述(由申请人提供)：人红细胞膜(RBCM)是研究最深入的质膜，不仅因为它是其他人类膜的可访问模型，还因为其组成部分的遗传和获得性缺陷会导致严重的病理。尽管进行了审查，但对区域协调机制的结构和功能的基本方面仍然知之甚少。在目标1中，我们将描述一种新发现的桥的结构和功能(带3到2-内收蛋白到血影蛋白)，它连接脂质双层和血影蛋白-肌动蛋白骨架。我们最近证明，带3的~1/3通过这个桥固定在光谱蛋白/肌动蛋白连接复合体上，桥的断裂会导致膜的断裂。为了评估该桥在体内的意义，我们将绘制内收蛋白在带3上的结合位置图，鉴定带3上阻止内收素结合的突变，产生包含突变带3的小鼠，并评估突变红细胞的形态和机械性能。作为这一目标的一个主要组成部分，描述了一种成像单个带3分子在完整红细胞中扩散的方法，作为一种灵敏地监测RBCM结构扰动的工具。带3的跨膜结构域(Msdb3)不仅介导阴离子跨膜转运，而且还组成一个跨膜蛋白复合体，包括血糖蛋白A、CD47、Rh蛋白、水通道蛋白和几种转运蛋白。为了在分子水平上了解msdb3的功能，我们将在高分辨率下求解它的晶体结构(这将是任何溶质载体IV类成员的第一个结构)。我们目前的晶体可以绕射到-lt；6e，虽然我们现在可以解决6e的结构，但我们有信心使用Aim 2中概述的新策略来产生更高分辨率的晶体。强有力的证据表明，1)红细胞新陈代谢，2)膜结构特性，3)离子传输受O2的调节。因为脱氧血红蛋白(而不是氧合Hb)与带3有很高的亲和力，而且带3又与负责上述每种性质的蛋白质结合，我们推测带3与脱氧Hb的可逆结合构成了红细胞膜氧合调节膜特性的“分子开关”。在目标3中，我们将使用最近发现的带3突变来验证这一假设：i)消除对脱氧Hb的所有亲和力，或ii)显著增强脱氧Hb对带3的亲和力，使得即使在饱和O2的情况下，脱氧Hb既不能释放其O2，也不能从带3解离。我们建议建立表达这两个突变条带3S的敲入小鼠，并使用小鼠来确定上述O2调节的属性是如预测的那样永久“开启”还是永久“关闭”。与公共健康相关：红细胞发挥着对我们的生存至关重要的各种功能，包括将氧气从肺部输送到组织，将二氧化碳从组织输送到肺部，传递一氧化氮以促进血液流动，参与血液凝结以促进伤口愈合，以及调节血液中发生的其他一些反应。我们的研究试图了解这些重要功能中每一个的分子基础，并在可能的情况下定义临床干预措施，以使上述过程发生故障的情况能够得到治疗。

DESCRIPTION (provided by applicant): The human erythrocyte membrane (RBCM) is the most thoroughly studied plasma membrane, not only because it serves as an accessible model of other human membranes, but also because inherited and acquired defects in its components lead to serious pathologies. Despite this scrutiny, fundamental aspects of the structure and function of the RBCM remain poorly understood. In Aim 1, we will characterize the structure and function of a newly discovered bridge (band 3 to 2-adducin to spectrin) that links the lipid bilayer to the spectrin-actin skeleton. We have recently demonstrated that ~1/3 of the band 3 population is anchored to the spectrin/actin junctional complex via this bridge and that rupture of the bridge leads to membrane fragmentation. To evaluate the significance of the bridge in vivo, we will map the binding site of adducin on band 3, identify mutations in band 3 that prevent adducin binding, generate a mouse containing the mutant band 3, and evaluate the morphological and mechanical properties of the mutant erythrocytes. Included as a major component of this aim is the characterization of a method to image the diffusion of single band 3 molecules in intact erythrocytes as a tool to sensitively monitor perturbations of RBCM structure. The membrane-spanning domain of band 3 (msdb3) not only mediates anion transport across the membrane, but also organizes a complex of membrane-spanning proteins, including glycophorin A, CD47, Rh proteins, aquaporin, and several transporters. In order to understand the function of msdb3 at a molecular level, we will solve its crystal structure at high resolution (This will be the first structure of any member of solute carrier class IV). We currently have crystals that diffract to <6E, and although we could solve a 6E structure now, we are confident that we can generate much higher resolution crystals using the novel strategies outlined in Aim 2. Strong evidence suggests that 1) red cell metabolism, 2) membrane structural properties, and 3) ion transport are regulated by O2. Because deoxyhemoglobin (but not oxyHb) binds with high affinity to band 3, and since band 3 associates with proteins responsible for each of the above properties, we hypothesize that the reversible association of band 3 with deoxyHb constitutes the "molecular switch" through which red cell oxygenation regulates membrane properties. In Aim 3, we will test this hypothesis using recently discovered band 3 mutations that either: i) eliminate all affinity for deoxyHb, or ii) enhance the affinity of deoxyHb for band 3 so significantly that deoxyHb can neither release its O2 nor dissociate from band 3, even at saturating O2. We propose to generate knock-in mice that express these two mutant band 3s, and use the mice to determine if the above O2- regulated properties are permanently "switched on" or permanently "switched off", as predicted. PUBLIC HEALTH RELEVANCE: The red blood cell performs a variety of functions critical to our survival, including transport of oxygen from the lungs to the tissues and carbon dioxide from the tissues to the lungs, delivery of nitric oxide to facilitate blood flow, participation in blood clotting to facilitate wound healing, and regulation of a number of other reactions that occur in the blood. Our research seeks to understand the molecular basis of each of these important functions, and where possible, to define clinical interventions that might enable treatment of conditions where the above processes malfunction.