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，不仅因为它可以作为其他人体膜的模型，而且因为其成分的遗传和获得性缺陷导致严重的病理。尽管进行了仔细审查，但对RBCM的结构和功能的基本方面仍然知之甚少。在目的1中，我们将描述一个新发现的桥（带3到2-内收蛋白到谱蛋白）的结构和功能，它将脂质双分子层连接到谱蛋白-肌动蛋白骨架。我们最近证明了约1/3的带3群通过这个桥固定在谱蛋白/肌动蛋白连接复合物上，并且桥的破裂导致膜断裂。为了评估该桥在体内的意义，我们将在3带上绘制内缩蛋白的结合位点，鉴定3带中阻止内缩蛋白结合的突变，生成含有突变3带的小鼠，并评估突变红细胞的形态学和力学特性。作为该目标的一个主要组成部分，本文描述了一种方法，该方法可以成像完整红细胞中单带3分子的扩散，作为一种敏感监测红细胞结构扰动的工具。带3的跨膜结构域（msdb3）不仅介导阴离子跨膜运输，而且还组织一个跨膜蛋白复合物，包括糖蛋白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，我们将以高分辨率解决其晶体结构（这将是溶质载流子IV类成员的第一个结构）。我们目前有衍射到<6E的晶体，尽管我们现在可以解决6E结构，但我们有信心使用Aim 2中概述的新策略可以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)膜结构特性和3)离子运输受O2调节。由于脱氧血红蛋白（而不是脱氧血红蛋白）与3号带具有高亲和力结合，并且3号带与负责上述每种特性的蛋白质结合，我们假设3号带与脱氧血红蛋白的可逆结合构成了“分子开关”，通过该开关，红细胞氧合调节膜的特性。在Aim 3中，我们将使用最近发现的3带突变来验证这一假设，这些突变要么：i)消除对deoxyHb的所有亲和力，要么ii)显著增强deoxyHb对3带的亲和力，以至于deoxyHb既不能释放其O2，也不能与3带分离，即使在饱和O2下。我们建议产生表达这两个突变带3s的敲入小鼠，并使用这些小鼠来确定上述O2调节特性是如预测的那样永久“开启”还是永久“关闭”。公共卫生相关性：红细胞对我们的生存具有多种至关重要的功能，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，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，确定临床干预措施，可能使上述过程出现故障的情况得到治疗。

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.