CYTOSKLETON PROTEINS & RED CELL VOLUME REGULATION--VOLUME SENSITIVE ION CHANNELS

(摘自申请者摘要)细胞体积调节是必要的 人红细胞(RBC)分化特征及疾病状态 有网织红细胞增多症和年轻细胞。镰状细胞(SS)、SC和CC 疾病，异常的体积调节导致细胞脱水和 多效性病理事件。不幸的是，RBC音量控制 人们对此知之甚少，特别是由于缺乏分子 鉴定出了负责体积调节的蛋白质，而且很少 了解这些蛋白质的调控机制。这个 这项提议的主要目标是从分子上定义红细胞体积- 敏感的离子转运体/通道蛋白和了解 这些蛋白质经历的结构和调控反应 音量正常。调查人员尤其对定义 细胞骨架在向离子传递体积信号中的作用 转运体/通道以及细胞骨架之间是否直接相互作用 蛋白质和这些蛋白质参与了继承人的调控。这些目标 将通过以下具体目标加以解决：(1)分子鉴定 负责红细胞体积敏感离子转运的蛋白质：pICln，K：c1 共转运体(K：c1-CT)和Na-K-c1共转运体(Na-K-2c1-CT)；以及 表达这些离子转运体/通道的克里特岛细胞和动物模型。 (2)确定带3低聚态在体积敏感信号中的作用 离子转运体/通道活性的转导和调节，以及测试 蛋白质磷酸化-去磷酸化是一种常见的假说 离子转运/通道调节的机制。如果是，请确定 磷酸化位点。(3)检验血红蛋白(Hb)的假设 与离子转运蛋白/通道的结合影响细胞骨架 蛋白质。明确细胞骨架调控细胞的机制 音量受到影响。(5)根据这些信息制定模型，并进行测试 红系细胞系中特定细胞骨架的模型 使用核酶和反义技术可以诱导缺陷。这个 研究应该大大增加对RBC体积的理解 监管。这些信息将允许制定针对以下目标的明智战略 防止红细胞脱水，这可能会对 对未来SS、SC和CC疾病的药物或基因治疗的影响。

(Adapted from Applicant's Abstract) Cell volume regulation is a necessary feature of human red cell (RBC) differentiation and in disease states with reticulocytosis and young cells. In sickle cell (SS), SC and CC disease, abnormal volume regulation leads to cell dehydration and pleiotropic pathological events. Unfortunately, RBC volume control is poorly understood, in particular, due to the lack of molecular identification of proteins responsible for volume regulation, and little understanding of the mechanisms by which these proteins are regulated. The major goals of this proposal are to molecularly define RBC volume- sensitive ion transporter/channel proteins and to understand the structural and regulatory responses these proteins undergo to restore normal volume. The investigators are particularly interested in defining the role of the cytoskeleton in transducing volume signals to the ion transporters/channel and whether direct interactions between cytoskeleton proteins and these proteins are involved int heir regulation. These goals will be addressed by the following specific aims: (1) Molecularly identify proteins responsible for RBC volume-sensitive ion transport: pICln, K:C1 co-transporter (K:C1-CT), and Na-K-C1 co-transporter (Na-K-2C1-CT); and crete cell and animal models expressing these ion transporters/channels. (2) Define the role of band 3 oligomeric state in volume-sensitive signal transduction and regulation of ion transporter/channel activity, and test the hypothesis that protein phosphorylation-dephosphorylation is a common mechanism for ion transport/channel regulation. If so, identify the phosphorylation sites. (3) Test the hypothesis that hemoglobin (Hb) binding to ion transport proteins/channels affects and cytoskeleton proteins. Define mechanisms by which cytoskeleton regulation of cell volume is affected. (5) Formulate models from this information, and test the models in erythroid cell lines where specific cytoskeleton deficiencies are induced using ribozyme and antisense technology. The studies should greatly increase the understanding the RBC volume regulation. This information will allow for sensible strategies aimed at preventing RBC dehydration, which would have a potentially significant impact on future drug or gene-based therapies for SS, SC, and CC diseases.