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共晶的调节特性 （RBC），在网状细胞/成熟的RBC过渡期间，以及鬼魂。 k- Cl Cotransport，一种抗ouabain的（OR）和严格依赖性的 当细胞肿胀或硫醇基团激活钾（K）转运蛋白 修改可能构成膜被动k的一小部分 渗透性。 K-CL Cotransport向外固执己见 绵羊和其他RBC的成熟RBC量减少，以及 在病理学上，患者的RBC脱水 血红蛋白病。 在绵羊中，k-cl共同运动发生在所有人中 低k（LK）类型的网状细胞和成熟的RBC中，但消失在 高k（HK）细胞，维持K-CL共转移的机制 然而，在LK中中等活性，在HK细胞中灭活，尚不清楚。 了解在此模型中k-cl共同竞争的过程 将检验以下假设：1。动力学和 K-CL共转运的热力学特性，最近阐明了 我们对于肿胀活化系统，无血红蛋白也很常见 鬼魂，以及网状细胞前体和成熟的RBC。 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测试。 K-CL共晶的动力学和热力学特性将是 与网状细胞中的或零型跨tans k涌入和排出相比 LK和HK基因型的成熟红色细胞，以及在重新密封的幽灵中 肿胀诱导的K-CL途径的途径。 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，已知引起激活或灭活的干预措施 转运蛋白。 膜结合的硫醇的存在对于K-至关重要 CL Cotransport功能将通过有选择性的无线电标签进行测试 受保护的硫醇和其他组，以及电泳验证 标记的膜成分以及通过定量相关性 同时测量的K-CL共晶剂固定示踪剂合并 活动。 了解该模型中运输变化的过程 会阐明为什么在人类RBC中纯合的血红蛋白S， K-CL共同竞争保持活跃，因此有助于不可逆 恶心。