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。 研究集中在KC 1 由细胞肿胀刺激的共转运系统。 的理解 这种转运系统的研究可能会导致镰状细胞的新疗法 疾病 兔红细胞用作模型系统，其中 膨胀刺激的K+通量很大，可以详细研究。 两 具体目标涉及运输机制。 测量 86 Rb+将用于测试特定的催化循环。 36 Cl-下的通量 将测量阴离子交换剂完全抑制的条件 以确定KCl共转运的化学计量。 大多数拟议的工作是为了调查的机制， 细胞体积增加25%可使KC 1增加10倍， 共运输通量 我们将使用一种新的方法， 测定并比较了KCl协同转运的激活和失活 一个简单的机械模型的预测。 以前的工作有 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 活化速率常数可检测地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值，低Mg++）也激活KC 1 共转运 运输随阶跃变化的速率 将测量这些参数以确定是否引起激活 活化速率常数、失活速率常数或 两者 基于最近对蛋白磷酸酶的研究， 抑制剂冈田酸是由 1型或（不太可能）2a型蛋白磷酸酶是必需的， 通过细胞肿胀激活KC 1共转运。 冈田酸敏感 兔红细胞中的蛋白磷酸酶将被表征为 测试的假设，激活的速率常数， 转运蛋白与特定的磷酸酶活性成比例（在 膜或细胞质）。 工作模型提出， 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测定，以检验 特定的激酶活性具有失活速率的特征， 恒定的运输。 除了研究负责的酶 为了调节KC 1共转运，将进行32 P磷酸化实验， 进行，试图确定底物是去磷酸化， 与运输的激活并行。

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.