Regulation of Erythrocyte Volume Homeostasis

项目摘要/摘要 PIEZO1最近被确定为哺乳动物机制中长期追逐的蛋白质。 感觉和拉伸激活的阳离子通道激活。我们发现PIEZO1基因突变导致 遗传性干细胞症，一种以红细胞脱水为特征的溶血性贫血，表明 PIEZO1在细胞体积动态平衡中起关键作用。PIEZO1是这一未知延伸的候选者- 诱导红细胞内钙激活的阳离子通路在红细胞衰老中起关键作用， 疟疾侵袭和循环压力。初步数据还表明，PIEZO1是一种优秀的 镰刀--一种脱氧诱导的未知阳离子通透途径--的候选基因 红细胞，在脱水级联反应开始时。因此，镰刀对于 镰状细胞病理学。尽管它很重要，但我们对控制PIEZO1的机制一无所知 在红系细胞中的表达、结构或功能。这项提案的总体目标是开始阐明 PIEZO1在红系细胞中调节和功能的分子机制。我们的初步研究 提示PIEZO1基因变异在遗传性干细胞症和镰状细胞病患者中存在，并且 这些变异与红细胞水合作用的改变有关。目标一的目标是认同 影响镰状细胞病患者红细胞水合作用的基因变异和数量增加 以及这些突变对PIEZO1表达、结构和功能的影响 功能。目标二的目标是建立遗传性干细胞症的小鼠模型，并分析 野生型和镰状细胞背景下体内HX相关Piezo1功能突变的获得。的目标是 目的3研究Piezo1功能丧失对野生型和非野生型红细胞功能的影响。 为了更好地了解调节红细胞体积稳态的分子机制， 解决了PIEZO1介导镰状细胞阳离子电流的假设，这种未确定的初始运输 镰状红细胞脱水的关键途径，并评估Piezo1缺乏对 镰状细胞病的参数。为了解决突变对PIEZO1结构和功能的影响， 基于功能细胞的PIEZO1膜蛋白表达、转运和电生理学分析 PIEZO1表达的新型体内稳定转染、单拷贝、可诱导的细胞模型将被执行。 将对转基因小鼠和受影响患者的成熟红细胞进行生理学研究 在各种细胞条件下执行。PIEZO1在许多细胞类型中都有表达，表明PIEZO1 可能在各种各样的细胞中调节重要的功能。因此，对红系细胞的研究可能会产生 可概括为许多关键的细胞过程或人类疾病的机械原理或生物学原理。

PROJECT SUMMARY/ABSTRACT PIEZO1 has recently been identified as the long sought after protein involved in mammalian mechano- sensation and stretch-activated cation channel activation. We have discovered mutations in PIEZO1 lead to hereditary xerocytosis, a hemolytic anemia characterized by primary erythrocyte dehydration, indicating PIEZO1 plays a critical role in cellular volume homeostasis. PIEZO1 is a candidate for the unidentified stretch- induced calcium-activated cation pathways in the red blood cell that play critical roles in erythrocyte aging, malaria invasion, and circulatory sheer stress. Preliminary data also indicate that PIEZO1 is an excellent candidate for Psickle, an unidentified cation permeability pathway induced by deoxygenation in sickle erythrocytes, at the initiation of the dehydration cascade. Therefore, Psickle is of fundamental importance to sickle cell pathobiology. Despite its importance, we have no knowledge of the mechanisms controlling PIEZO1 expression, structure, or function in erythroid cells. The overall goal of this proposal is to begin to elucidate the molecular mechanisms involved in PIEZO1 regulation and function in erythroid cells. Our preliminary studies indicate that variants of the PIEZO1 gene occur in hereditary xerocytosis and sickle cell disease patients and that these variants are associated with alterations in erythrocyte hydration. The goal of aim one is identification of genetic variants influencing erythrocyte hydration in patients with sickle cell disease and increased numbers of dense cells and characterization of the effect of these mutations on PIEZO1 expression, structure and function. The goal of aim two is to create a murine model of hereditary xerocytosis and analyze the influence of HX-associated Piezo1 gain of function mutations in vivo on wild type and sickle cell backgrounds. The goal of aim 3 is the characterization of the influence of Piezo1 loss of function on erythrocyte function in wild type and SCD erythrocytes to better understand molecular mechanisms regulating erythrocyte volume homeostasis, to address the hypothesis that PIEZO1 mediates the cation currents of Psickle, the unidentified, incipient transport pathway critical for dehydration of sickle erythrocytes, and to assess the influence of Piezo1 deficiency on parameters of sickle cell disease. To address the influence of mutations on PIEZO1 structure and function, functional cell-based assays of PIEZO1 membrane protein expression, trafficking, and electrophysiology in a novel, in vivo stably-transfected, single-copy, inducible cell model of PIEZO1 expression will be performed. Physiologic studies of mature erythrocytes from genetically modified mice and affected patients will be performed under a variety of cellular conditions. PIEZO1 is expressed in many cell types, indicating PIEZO1 likely mediates important functions in a wide variety of cells. Thus studies in erythroid cells may yield mechanistic or biological principles generalizable to many critical cellular processes or human diseases.