Regulation of Erythrocyte Volume Homeostasis

描述（由申请方提供）：水和溶质稳态的维持对红细胞的存活至关重要。红细胞水化的原发性疾病是一组遗传性疾病，范围从脱水到过度水化的细胞。根据容量稳态的扰动程度，可能导致溶血性贫血。当细胞水合作用的扰动与另一种状况相关时，发生红细胞水合作用的继发性疾病，例如通常伴随镰状细胞病或β血红蛋白病的脱水。在继发性疾病中，改变的红细胞水合作用可能是疾病病理学的主要贡献者。PIEZO 1最近被鉴定为长期以来一直在寻找的蛋白质，参与哺乳动物的机械感觉和牵张激活的阳离子通道激活。我们发现PIEZO 1突变导致遗传性干细胞症（HX），这是一种以原发性红细胞脱水为特征的溶血性贫血，表明PIEZO 1在细胞体积稳态中起重要作用。PIEZO 1是红细胞中未鉴定的牵张诱导阳离子途径的候选者，其在红细胞老化、疟疾侵袭和循环切应力中起关键作用。PIEZO 1也是Psickle的极好候选者，Psickle是镰状红细胞中在对镰状细胞病理学具有根本重要性的脱水级联起始时的未鉴定的阳离子渗透性途径。尽管其重要性，我们没有知识的机制控制PIEZO 1的表达，调节，结构或功能，其在调节红系细胞的体积稳态的作用。拟议的研究结合联合收割机最先进的细胞，遗传，蛋白质组学和生理学技术，以创新的，多学科的方式表征PIEZO 1的表达，结构和功能。研究包括在PIEZO 1表达的新型体内稳定转染的单拷贝诱导型cII模型中PIEZO 1膜蛋白表达、运输和电生理学的功能性、基于细胞的测定。将创建和表征新的Piezo1基因修饰小鼠模型，包括HX小鼠模型。最后，定量的基于MRM的蛋白质组学研究和国家的最先进的机械转导生理技术将被应用于各种细胞条件下的HX患者的红细胞。PIEZO 1存在于许多细胞类型中，包括淋巴细胞、内皮细胞、肾细胞和神经细胞，表明它可能在多种细胞中介导重要功能。因此，在红系细胞中的研究可能产生可推广到许多关键细胞过程或人类疾病的机械或生物学原理。

DESCRIPTION (provided by applicant): Maintenance of water and solute homeostasis is critical to survival of the erythrocyte. Primary disorders of erythrocyte hydration are a group of inherited disorders ranging from dehydrated to overhydrated cells. Depending on the degree of perturbation of volume homeostasis, hemolytic anemia may result. Secondary disorders of erythrocyte hydration occur when perturbation in cell hydration is associated with another condition, for instance the dehydration that commonly accompanies sickle cell disease or beta hemoglobinopathies. In secondary disorders, altered erythrocyte hydration may be a major contributor to disease pathology. PIEZO1 has recently been identified as the long sought after protein involved in mammalian mechanosensation and stretch-activated cation channel activation. We have discovered mutations in PIEZO1 that lead to hereditary xerocytosis (HX), a hemolytic anemia characterized by primary erythrocyte dehydration, indicating PIEZO1 plays an important role in cellular volume homeostasis. PIEZO1 is a candidate for unidentified stretch-induced cation pathways in the erythrocyte that play critical roles in erythrocyte aging, malaria invasion, and circulatory sheer stress. PIEZO1 is also an excellent candidate for Psickle, an unidentified cation permeability pathway in sickle erythrocytes at the initiation of the dehydratio cascade of fundamental importance to sickle cell pathobiology. Despite its importance, we have no knowledge of the mechanisms controlling PIEZO1 expression, regulation, structure or function and its role in regulation of volume homeostasis in erythroid cells. The proposed studies combine state of the art cellular, genetic, proteomic, and physiologic technologies to characterize the expression, structure and function of PIEZO1, in an innovative, multidisciplinary manner. Studies include functional, cell-based assays of PIEZO1 membrane protein expression, trafficking, and electrophysiology in a novel, in vivo stably-transfected, single-copy, inducible cll model of PIEZO1 expression. New genetically modified murine models of Piezo1, including a murine model of HX, will be created and characterized. Finally, quantitative MRM-based proteomic studies and state-of-the-art mechanotransduction physiologic techniques will be applied to erythrocytes from HX patients under a variety of cellular conditions. PIEZO1 is found in many cell types including lymphocytes, endothelial, kidney, and neural cells, indicating it likey 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.