MEL CELL K/CL COTRANSPORT--CHARACTERIZATION AND CLONING

MEL 细胞 K/CL 协同转运——表征和克隆

• 批准号: 2225382
• 负责人: Eric J Delpire
• 金额: $ 11.25万
• 依托单位: BRIGHAM AND WOMEN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-08-01 至 1996-09-16
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2225382
• 关键词: Xenopus oocyte; alternatives to animals in research; cell differentiation; cell morphology; chloride channels; complementary DNA; erythrocyte membrane; erythroleukemia; gene expression; genetic regulation; ion transport; laboratory mouse; membrane activity; membrane transport proteins; messenger RNA; molecular cloning; potassium channel; site directed mutagenesis; sodium potassium exchanging ATPase; tissue /cell culture; trout /salmon

In recent years, a number of investigations carried out on the passive ion permeability of erythrocyte membranes have revealed that a fraction of the ouabain-resistant K+ fluxes is insensitive to Na+ and depends on the presence of Cl-. The transport mechanism underlying the ouabain-, and Na+-insensitive, Cl--dependent K+ flux has been defined as the K-Cl cotransporter. Due to the direction of KCI gradients, K-Cl cotransport when activated induces a loss of KCI and a cell volume reduction. Since a reduction in cell volume is a well established event in red blood cell maturation and K-Cl fluxes through the K-Cl cotransporter are high in reticulocytes and almost absent in mature erythrocytes, it has been hypothesized that the pathway plays a significant role in the maturation process. The kinetic details and the regulation of the pathway have been extensively studied these last few years, but its molecular identity is still unknown. This proposal targets two major objectives: 1) To test the hypothesis that K-Cl cotransport participates in the cell volume reduction occurring through the overall maturation process. We propose to characterize the role of Cl--dependent K+ transport together with other K+ transport systems from undifferentiated cells to reticulocytes by using a mouse erythroleukemia (MEL) cell line (nucleated as opposed to mature mammalian red cells). 2) To clone and identity the transport protein which mediates the coupled K-Cl fluxes. Since trout hepatocytes present the highest level of K-Cl cotransport activity, and since total RNA isolated from trout liver and injected to Xenopus oocytes lead to expression of a Cl-dependent K+ flux, we propose to identity the trout liver cDNA encoding the K-Cl cotransporter. Once this cDNA clone is isolated, we propose to clone the mammalian homologue and initiate a comparative study of mRNA levels, K-Cl cotransport activity, and regulation in the different stages of MEL cell differentiation. Finally, a structure/function study is proposed to uncover the sites of ion binding and to determine whether the N-ethylmaleimide activation occurs through binding to the transport molecule or not. Results of these studies would explain part of the physiological mechanisms leading to the recruitment and/or disappearance of K-Cl cotransport units, or leading to the activation/inactivation process occurring during red cell maturation. Finally, these studies will shed some light on the abnormal activation of this transport pathway in mature human red cells containing hemoglobin S or C.

近年来，对被动进行了许多调查 红细胞膜的离子渗透性显示了一部分 抗ouabain耐药的K+通量对Na+不敏感，取决于 Cl-的存在。 Ouabain-和 Na+不敏感的Cl-依赖性K+通量已定义为K-CL 共转运者。由于KCI梯度的方向，K-CL共晶型 当激活时，会诱导KCI的损失和细胞体积减少。自从 细胞体积减少是红细胞中良好的事件 通过K-CL共转运蛋白的成熟和K-CL通量很高 在成熟的红细胞中，网状细胞几乎不存在，已经是 假设该途径在成熟中起着重要作用 过程。动力学细节和途径的调节 最近几年对其分子身份进行了广泛的研究 仍然未知。该建议针对两个主要目标：1）测试 假设K-CL共同运动参与细胞体积 通过整体成熟过程减少。我们建议 表征Cl-依赖性K+传输的作用 其他K+运输系统从未分化的细胞到网状细胞 通过使用小鼠红血病（MEL）细胞系（成核与相反 到成熟的哺乳动物红细胞）。 2）克隆并识别运输 介导耦合K-CL通量的蛋白质。自鳟鱼肝细胞以来 提出最高水平的K-CL共同竞技运动，并且由于总计 从鳟鱼肝分离并注入爪蟾卵母细胞的RNA导致 依赖Cl依赖的K+通量的表达，我们提出要识别鳟鱼 编码K-CL共转运蛋白的肝cDNA。一旦这个cDNA克隆是 孤立，我们建议克隆哺乳动物的同源物并启动A mRNA水平，K-CL共转运活性和 在MEL细胞分化的不同阶段进行调节。最后， 提出了一项结构/功能研究以发现离子的位置 结合并确定是否发生N-乙基甲米酰亚胺激活 通过与运输分子结合。这些结果 研究将解释一部分生理机制 K-CL共同运动单位的招聘和/或失踪或领导 在红细胞期间发生的激活/灭活过程 成熟。最后，这些研究将使异常有所了解 在含有成熟的人类红细胞中的该传输途径的激活 血红蛋白S或C。