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

（改编自申请人的摘要）细胞体积调节是必要的 人红细胞（RBC）分化的特征和疾病状态 网织红细胞增多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， 多效性病理事件。不幸的是，RBC体积控制 特别是由于缺乏分子生物学， 鉴定负责体积调节的蛋白质， 了解这些蛋白质的调节机制。的 该提案的主要目标是从分子上定义RBC体积- 敏感的离子转运蛋白/通道蛋白，并了解 这些蛋白质经历的结构和调节反应， 正常音量。研究人员特别感兴趣的是， 细胞骨架在将体积信号转导到离子中的作用 转运蛋白/通道以及细胞骨架之间是否直接相互作用 蛋白质，这些蛋白质参与其调节。这些目标 将通过以下具体目标来解决：（1）分子鉴定 负责红细胞体积敏感性离子转运的蛋白质：pICln，K：C1 共转运蛋白（K：C1-CT）和Na-K-C1共转运蛋白（Na-K-2C 1-CT）;和 表达这些离子转运蛋白/通道的Crete细胞和动物模型。 (2)定义带3寡聚状态在体积敏感信号中的作用 离子转运蛋白/通道活性的转导和调节，以及测试 假设蛋白质磷酸化-去磷酸化是一种常见的 离子转运/通道调节机制。如果是，请确定 磷酸化位点。(3)检验血红蛋白（Hb） 与离子转运蛋白/通道结合影响细胞骨架 proteins.定义细胞骨架调节细胞的机制 量受到影响。(5)根据这些信息制定模型，并测试 红细胞系中的模型， 使用核酶和反义技术诱导缺陷。的 研究应该大大增加对红细胞体积的理解 调控这些信息将有助于制定明智的战略， 防止红细胞脱水，这将有一个潜在的显着 对SS、SC和CC疾病未来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.