KINETICS AND REGULATION OF ERYTHROCYTE K-CL COTRANSPORT

这个项目提出了一项研究，包括动力学、热力学和 绵羊红细胞钾-氯共转运的调节特性 (红细胞)，在网织红细胞/成熟红细胞过渡期间，以及在幽灵中。K- 氯共转运，一种抗哇巴因(OR)和严格依赖氯的 钾(K)转运体，当被细胞膨胀或硫醇基团激活时 修饰，可能构成膜的被动K的主要部分 渗透性。表面上看，K-Cl共转运被牵连到 绵羊和其他红细胞的成熟红细胞体积减少，以及 某些患者红细胞脱水的病理变化 血红蛋白病。在绵羊体内，钾-氯共转运发生在 网织红细胞和成熟的低K(LK)型红细胞，但在 高K(HK)细胞，维持K-Cl共转运的机制 然而，在LK中适度活跃，在HK细胞中失活，目前尚不清楚。 了解该模型中钾-氯共转运的内涵化过程 将检验以下假设：1.动力学和 钾-氯共转运的热力学特征，最近由 我们对于肿胀激活系统，也是常见的无血红蛋白 幽灵，网织红细胞前体和成熟红细胞。2. 监管涉及运输器的膜组件以及 细胞质因子。这些一般性假设将按如下方式进行检验。 钾-氯共转运的动力学和热力学性质将是 网织红细胞和网织红细胞中或零-反式钾流入和流出的比较 LK和HK两种基因型的成熟红细胞，以及重新密封的幽灵 肿胀诱导的K-Cl途径。钾-氯的调节 将研究具有不同细胞内镁的LK细胞中的共转运， 阴离子和Ph，已知可导致激活或失活的干预 传送器的。膜结合硫醇的存在对K- CL共转运功能将通过选择性地进行放射性标记来测试 受保护的硫醇和其他基团，以及电泳法验证 标记的膜组分，以及通过对 同时测定K-Cl共转运蛋白的示踪剂掺入 活动。了解该模型中运输变化的过程 例如，将解释为什么在人类红细胞中存在血红蛋白纯合S， 钾-氯共转运仍然活跃，因此有助于不可逆 像镰刀一样。

This project proposes a study of the kinetic, thermodynamic and regulatory properties of K-Cl cotransport of sheep red blood cells (RBCs), during the reticulocyte/mature RBC transition, and in ghosts. K- Cl cotransport, a ouabain-resistant (OR) and strictly Cl-dependent potassium (k) transporter, when activated by cell swelling or thiol group modification, may constitute a major fraction of the membrane's passive K permeability. Outwardly poised, K-Cl cotransport has been implicated in maturational RBC volume reduction of sheep and other RBCs, and pathologically in RBC dehydration of patients with certain hemoglobinopathies. In sheep, K-Cl cotransport occurs in all reticulocytes and in mature RBCs of the low K (LK) type but disappears in high K (HK) cells, The mechanism by which K-Cl cotransport is maintained moderately active in LK, however, inactivated in HK cells, is unknown. To understand the process of K-Cl cotransport involution in this model the following hypothesis will be tested: 1. The kinetic and thermodynamic characteristics of K-Cl cotransport, recently elucidated by us for the swelling activated system, are also common to hemoglobin-free ghosts, and to both reticulocyte precursor and mature RBCs. 2. Regulation involves membrane components of the transporter, as well as cytoplasmic factors. These general hypotheses will be tested as follows. The kinetic and thermodynamic properties of K-Cl cotransport will be compared by OR zero-trans K influxes and effluxes in reticulocytes and mature red cells of both LK and HK genotypes, and in resealed ghosts with those of the swelling induced K-Cl pathway. The regulation of K-Cl cotransport will be studied in LK cells with varied intracellular Mg, anions, and Ph, interventions known to cause activation or inactivation of the transporter. The presence of membrane-bound thiols crucial for K- Cl cotransport function will be tested by radio-labelling of selectively protected thiols and other groups, and electrophoretic verification of labelled membrane components, as well as by quantitative correlation of tracer incorporation with simultaneously measured K-Cl cotransprot activity. Understanding the process of transport changes in this model will elucidate why for example in human RBCs homozygous for hemoglobin S, K-Cl cotransport remains active and thus contributes to irreversible sickling.