ERYTHROCYTE POTASSIUM TRANSPORT AND VOLUME REGULATION

红细胞钾转运和容量调节

• 批准号: 2218478
• 负责人: MICHAEL L JENNINGS
• 金额: $ 7.55万
• 依托单位: UNIVERSITY OF TEXAS MED BR GALVESTON
• 依托单位国家: 美国
• 财政年份: 1987
• 资助国家: 美国
• 起止时间: 1987-09-30 至 1995-09-14
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2218478
• 关键词: cell morphology; chlorine; disease /disorder model; edema; enzyme inhibitors; erythrocytes; laboratory rabbit; membrane transport proteins; okadaic acid; phosphoprotein phosphatase; phosphorylation; potassium; sickle cell anemia; stoichiometry

The long-term objective of this work is to understand the mechanism of cell volume regulation in mammalian red blood cells. The studies focus on a KC1 cotransport system that is stimulated by cell swelling. An understanding of this transport system could lead to new therapies for sickle cell disease. Rabbit red cells are used as a model system in which the swelling-stimulated K+ flux is large and can be studied in detail. Two of the specific aims concern the mechanism of transport. Measurements of 86Rb+ will be used to test specific catalytic cycles. Fluxes of 36Cl-under conditions of complete inhibition of the anion exchanger will be measured to determine the stoichiometry of KC1 cotransport. Most of the proposed work is designed to investigate the mechanism by which a 25% increase in cell volume can produce a 10-fold increase in the KC1 cotransport flux. We will use a new approach in which the rates of activation and inactivation of KC1 cotransport are measured and compared with the predictions of a simple mechanistic model. Previous work had shown that the rate constant for inactivation of transport is strongly volume dependent. Further experiments will determine whether the activation rate constant is detectably affected by cell volume. Besides cell swelling, two other interventions (low pH, low Mg++) also activate KC1 cotransport. The rates of change of transport following step changes in these parameters will be measured to determine whether activation is caused by a change in the activation rate constant, inactivation rate constant, or both. The working hypothesis, based on recent work with the protein phosphatase inhibitor, okadaic acid, is that a net dephosphorylation catalyzed by either type 1 or (less likely) type 2a protein phosphatase is necessary for the activation of KC1 cotransport by cell swelling. Okadaic acid-sensitive protein phosphatases in rabbit red blood cells will be characterized to test the hypothesis that the rate constant for activation of the transporter is proportional to a particular phosphatase activity (in either membrane or cytoplasm). The working model proposes that transport is activated by net dephosphorylation caused mainly by protein kinase inhibition rather than phosphatase activation. Protein kinase activities will be determined in membrane and cytoplasm to test the hypothesis that a particular kinase activity has the characteristics of the inactivation rate constant for transport. In addition to studying the enzymes responsible for regulation of KC1 cotransport, 32P phosphorylation experiments will be conducted to try to identify the substrate that is dephosphorylated in parallel with activation of transport.

这项工作的长期目标是了解细胞的机制 哺乳动物红细胞的容量调节。 研究重点是 KC1 受细胞肿胀刺激的协同转运系统。 一种理解 这种运输系统的研究可能会带来镰状细胞的新疗法 疾病。 兔红细胞被用作模型系统，其中 膨胀刺激的 K+ 通量很大，可以详细研究。 两个 具体目标涉及运输机制。 测量值 86Rb+ 将用于测试特定的催化循环。 36Cl以下的通量 将测量阴离子交换剂完全抑制的条件 确定 KC1 协同转运的化学计量。 大部分拟议的工作旨在研究机制 细胞体积增加 25% 可使 KC1 增加 10 倍 共转运通量。 我们将使用一种新方法，其中 测量并比较 KC1 共转运的激活和失活 与简单机械模型的预测。 之前的工作有 表明运输失活的速率常数强烈 体积相关。 进一步的实验将确定是否 激活速率常数明显受到细胞体积的影响。 除了 细胞肿胀，另外两种干预措施（低 pH、低 Mg++）也激活 KC1 协同运输。 运输的变化率随着步骤的变化而变化 将测量这些参数以确定是否引起激活 通过激活速率常数、失活速率常数的变化，或 两个都。 工作假设基于最近对蛋白磷酸酶的研究 抑制剂，冈田酸，是一种由以下物质催化的净去磷酸化作用： 1 型或（不太可能）2a 型蛋白磷酸酶对于 通过细胞肿胀激活 KC1 共转运。 冈田酸敏感 兔红细胞中的蛋白磷酸酶将被表征为 检验以下假设：激活的速率常数 转运蛋白与特定的磷酸酶活性成正比（在任一 膜或细胞质）。 该工作模型提出，运输是 主要由蛋白激酶引起的净去磷酸化激活 抑制而不是磷酸酶激活。 蛋白激酶活性 将在膜和细胞质中测定，以检验以下假设： 特定激酶活性具有失活率的特点 运输常数。 除了研究负责的酶之外 为了调节 KC1 共转运，32P 磷酸化实验将 试图鉴定去磷酸化的底物 与运输激活同时进行。