ERYTHROCYTE POTASSIUM TRANSPORT AND VOLUME REGULATION

这项工作的长期目标是了解细胞的机制 哺乳动物红细胞的体积调节。研究的重点是Kc1 由细胞肿胀刺激的共运输系统。一种理解 这种运输系统的研究可能会导致镰状细胞的新疗法 疾病。以兔红细胞为模型系统，建立了兔红细胞模型。 膨胀刺激的K+通量大，可以进行详细的研究。其中两个 具体目标涉及运输机制。测量 86Rb+将用于测试特定的催化循环。~(36)Cl-在空气中的通量 将测量完全抑制阴离子交换器的条件 测定Kc1共转运的化学计量学。 大多数拟议的工作都是为了研究 细胞体积增加25%可以使Kc1增加10倍 共传输通量。我们将使用一种新的方法，在这种方法中 测定和比较了Kc1共转运蛋白的激活和失活 与一个简单的机械模型的预测。以前的工作有 表明了运输失活的速率常数是很强的 取决于音量。进一步的实验将确定是否 活化速率常数明显受细胞体积的影响。此外 另外两种干预措施(低pH、低镁离子)也能激活Kc1 联运公司。运输的变化率随着以下步骤的变化 将测量这些参数以确定是否导致激活 通过改变激活速率常数、失活速率常数或 两者都有。 工作假说，基于最近对蛋白质磷酸酶的研究 抑制剂冈田酸是一种由其催化的净脱磷 类型1或(可能性较小)类型2a蛋白磷酸酶是必需的 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 检验这样的假设：激活的速率常数 转运蛋白与特定的磷酸酶活性成正比(在 膜或细胞质)。工作模式提出，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 将在细胞膜和细胞质中确定，以检验一种 特定的激酶活性具有失活率的特征 用于运输的常量。除了研究负责的酶 对于Kc1共转运的调节，将进行32P磷酸化实验 进行以尝试识别脱磷酸化的底物 与运输的激活同时进行。

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.