KINETICS AND REGULATION OF ERYTHROCYTE K-CL COTRANSPORT

红细胞 K-CL 协同转运的动力学和调节

• 批准号: 2139988
• 负责人: PETER K LAUF
• 金额: $ 15.9万
• 依托单位: WRIGHT STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1985
• 资助国家: 美国
• 起止时间: 1985-09-01 至 1997-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2139988
• 关键词: acid base balance; adenosine triphosphate; atomic absorption spectrometry; bicarbonates; cell morphology; chemical kinetics; chlorine; electrophoresis; erythrocyte membrane; erythrocytes; genotype; ion transport; ionophores; magnesium; maleimides; mathematical model; membrane activity; membrane permeability; membrane transport proteins; nuclear magnetic resonance spectroscopy; nystatin; potassium; protein structure function; radiotracer; reticulocytes; sheep; thermodynamics; thiols

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.

该项目提出了动力学、热力学和 绵羊红细胞 K-Cl 共转运的调控特性 （红细胞），在网织红细胞/成熟红细胞转变期间，以及在鬼影中。 K- Cl 协同转运，具有哇巴因抗性 (OR) 且严格依赖 Cl 钾 (k) 转运蛋白，当被细胞肿胀或硫醇基激活时 修饰，可能构成膜被动 K 的主要部分 渗透性。 表面上平静的 K-Cl 协同转运与 绵羊和其他红细胞的成熟红细胞体积减少，以及 患有某些疾病的患者红细胞脱水的病理学 血红蛋白病。 在绵羊中，K-Cl 共转运发生在所有部位 网织红细胞和低 K (LK) 型的成熟红细胞中，但在 高 K (HK) 细胞，维持 K-Cl 共转运的机制 在 LK 中具有中等活性，但在 HK 细胞中失活尚不清楚。 了解该模型中 K-Cl 共转运内合的过程 将检验以下假设： 1. 动力学和 K-Cl 共转运的热力学特征，最近由 我们用于肿胀激活系统，也常见于无血红蛋白 鬼影，以及网织红细胞前体和成熟红细胞。 2. 调节涉及转运蛋白的膜成分，以及 细胞质因子。 这些一般假设将进行如下检验。 K-Cl 共转运的动力学和热力学性质为 通过 OR 零反式 K 流入和流出网织红细胞和 LK 和 HK 基因型的成熟红细胞，以及重新密封的鬼魂 肿胀诱导的 K-Cl 途径的那些。 K-Cl的调节 将在具有不同细胞内 Mg 的 LK 细胞中研究共转运， 阴离子和 Ph，已知导致激活或失活的干预措施 运输商的。 膜结合硫醇的存在对于 K-至关重要 Cl共转运功能将通过选择性标记的放射性标记进行测试 受保护的硫醇和其他基团，以及电泳验证 标记的膜成分，以及通过定量相关 示踪剂掺入同时测量的 K-Cl 共转运 活动。 了解该模型中运输变化的过程 将阐明为什么例如在血红蛋白 S 纯合的人类红细胞中， K-Cl 共转运保持活跃，因此有助于不可逆 镰刀。