Studies of Erythrocyte Membrane Structure

描述（由申请人提供）：人红细胞膜（RBCM）是研究最彻底的质膜，不仅因为它是其他人体膜的可访问模型，还因为其组分中的遗传性和获得性缺陷导致严重的病理学。尽管进行了这种审查，但对管理区域局的结构和职能的基本方面仍然知之甚少。在目标1中，我们将表征新发现的连接脂双层与血影蛋白-肌动蛋白骨架的桥梁（带3至2-内收蛋白至血影蛋白）的结构和功能。我们最近已经证明，约1/3的带3人口通过这座桥锚定到血影蛋白/肌动蛋白连接复合物，桥的断裂导致膜断裂。为了评估体内桥的意义，我们将绘制带3上内收蛋白的结合位点，鉴定带3中阻止内收蛋白结合的突变，产生含有突变带3的小鼠，并评估突变红细胞的形态学和机械特性。包括作为一个主要组成部分，这一目标是表征的方法，以图像的扩散的单一带3分子在完整的红细胞作为一种工具，灵敏地监测扰动的红细胞膜结构。带3的跨膜结构域（msdb 3）不仅介导阴离子跨膜转运，而且组织跨膜蛋白的复合物，包括血型糖蛋白A、CD 47、Rh蛋白、水通道蛋白和几种转运蛋白。为了在分子水平上理解msdb 3的功能，我们将以高分辨率解析其晶体结构（这将是溶质载体IV类任何成员的第一个结构）。我们目前有晶体的分辨率<6 E，虽然我们现在可以解决6 E结构，但我们相信我们可以使用目标2中概述的新策略生成更高分辨率的晶体。强有力的证据表明，1）红细胞代谢，2）膜结构特性，3）离子转运受O2调节。由于脱氧血红蛋白（但不是oxyHb）结合带3具有高亲和力，并由于带3与负责上述每一个属性的蛋白质，我们假设，可逆的协会带3与脱氧血红蛋白构成的“分子开关”，通过红细胞氧合调节膜的性能。在目标3中，我们将使用最近发现的带3突变来检验这一假设，所述带3突变：i）消除对脱氧血红蛋白的所有亲和力，或ii）显著增强脱氧血红蛋白对带3的亲和力，使得脱氧血红蛋白既不能释放其O2也不能从带3解离，即使在饱和O2下。我们建议产生敲入小鼠表达这两个突变体带3，并使用小鼠来确定上述O2调节特性是否永久“打开”或永久“关闭”，如预测的那样。公共卫生相关性：红细胞执行对我们的生存至关重要的各种功能，包括将氧气从肺输送到组织，将二氧化碳从组织输送到肺，输送一氧化氮以促进血液流动，参与血液凝固以促进伤口愈合，以及调节血液中发生的许多其他反应。我们的研究旨在了解这些重要功能的分子基础，并在可能的情况下，确定可能能够治疗上述过程发生故障的条件的临床干预措施。

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.