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: 3474043
• 负责人: Eric J Delpire
• 金额: $ 13.15万
• 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 1998-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3474043
• 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，针对被动式的研究开展了多项研究。 红细胞膜的离子渗透性表明，一小部分 耐哇巴因的 K+ 通量对 Na+ 不敏感，并且取决于 Cl-的存在。哇巴因的运输机制，和 Na+ 不敏感、Cl 依赖的 K+ 通量被定义为 K-Cl 协同转运蛋白。由于 KCI 梯度的方向，K-Cl 共转运 当激活时会导致 KCI 损失和细胞体积减少。自从 细胞体积减少是红细胞中一个已确定的事件 通过 K-Cl 协同转运蛋白的成熟和 K-Cl 通量高 网织红细胞，成熟红细胞中几乎不存在， 假设该途径在成熟过程中发挥重要作用 过程。该途径的动力学细节和调控已被 过去几年进行了广泛的研究，但其分子身份是 仍然未知。该提案有两个主要目标：1）测试 K-Cl 共转运参与细胞体积的假设 整个成熟过程中发生的减少。我们建议 表征 Cl- 依赖性 K+ 运输的作用以及 从未分化细胞到网织红细胞的其他 K+ 转运系统 通过使用小鼠红白血病 (MEL) 细胞系（有核细胞，而不是 成熟的哺乳动物红细胞）。 2) 克隆和识别传输 介导耦合 K-Cl 通量的蛋白质。由于鳟鱼肝细胞 呈现最高水平的 K-Cl 协同转运活性，并且由于总 从鳟鱼肝脏中分离出的 RNA 注射到非洲爪蟾卵母细胞中 Cl 依赖性 K+ 通量的表达，我们建议识别鳟鱼 编码 K-Cl 协同转运蛋白的肝脏 cDNA。一旦这个 cDNA 克隆被 分离后，我们建议克隆哺乳动物同源物并启动 mRNA 水平、K-Cl 共转运活性和 MEL 细胞分化不同阶段的调控。最后， 提议进行结构/功能研究以揭示离子的位点 结合并确定是否发生 N-乙基马来酰亚胺活化 通过或不与运输分子结合。这些结果 研究将解释导致这种现象的部分生理机制 K-Cl 共转运单位的招募和/或消失，或导致 红细胞期间发生的激活/失活过程 成熟。最后，这些研究将为异常现象提供一些线索 成熟人红细胞中该转运途径的激活含有 血红蛋白 S 或 C。