CYTOSOLIC MODULATION OF PLASMA MEMBRANE ION TRANSPORT

质膜离子运输的细胞质调节

• 批准号: 3236472
• 负责人: MARK A MILANICK
• 金额: $ 8.13万
• 依托单位: UNIVERSITY OF MISSOURI-COLUMBIA
• 依托单位国家: 美国
• 财政年份: 1986
• 资助国家: 美国
• 起止时间: 1986-08-01 至 1997-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3236472
• 关键词: adenosinetriphosphatase; calcium; calcium metabolism; calmodulin; cell differentiation; cell membrane; chemical structure function; conformation; erythrocytes; ferrets; heart failure; hypertension; ion transport; membrane potentials; membrane structure; membrane transport proteins; muscle contraction; muscular dystrophy; potassium; sickle cell anemia

The long-term objectives of this research are to elucidate some of the mechanisms of ion transport, to determine how changes of the cellular environment alter transport rate. The focus of this project is the calmodulin activated Ca pump of human red cells. The Ca pump offers an unusual opportunity for structure-function studies; a portion of the C terminal of the pump, binds to another portion of the pump to inhibit transport. The red cell Ca pump is also a model for plasma membrane Ca pumps from other cells. Experiments on similar modes of operation of the Na pump are planned in order to provide a basis for a comparison between these two pumps and to determine if the mechanism is conserved. The specific aims are to: I. examine other modes of operation. In particular to compare the modulation of the rate of Na pump mediated ATP/ADP exchange and Na/Na exchange with the analogous modes of the Ca pump. These modes provide information about the order of ligand binding and release during the steady state operation of the Na (or Ca) portion of the pump cycle. II. characterize chemical modifiers and inhibitors. In particular to determine the relation between the eosin inhibition site, the FITC site and the ATP site. III. characterize auto-inhibitory peptide interactions. In particular to determine some electrostatic and steric constraints on the interaction between the C-terminal auto-inhibitory peptide, C20W, and the proteolysis-activated Ca pump. These 3 complimentary approaches will provide a foundation for evaluation of future chemical and genetic manipulations. For example, the conformations involved in Ca/Ca exchange may reduce the reactivity or accessibility of the lysine with which FITC reacts. The experimental protocol is to examine the effect of ligands, inhibitors and peptides on Ca uptake into inside out vesicles made from human red blood cells and to examine other modes of operation of the Ca pump. A transient rise in Ca is important for signalling in such processes as muscle contraction, cell differentiation and cell proliferation. The Ca pump restores Ca to basal levels and therefore assists in terminating the signal. Defects in Ca pump activity would lead to an increase in Ca with subsequent alterations of key cytoplasmic functions. Such ionic alterations have been implicated in several disease states including renal and cardiac failure, hypertension, and muscular dystrophy. Alterations of the red cell Ca pump have been reported in sickle cell disease.

这项研究的长期目标是阐明一些 离子运输机制，以确定细胞的变化， 环境改变运输速率。 该项目的重点是 钙调素激活人红细胞钙泵。 钙泵提供了 结构-功能研究的不寻常机会; C的一部分 泵的另一部分结合到泵的另一部分，以抑制 运输 红细胞钙泵也是质膜钙泵的模型 其他细胞的泵。 在类似操作模式下的实验 钠泵的计划，以提供一个基础，比较 这两个泵，并确定是否该机制是保守的。 具体目标是： I.检查其他操作模式。 特别是比较 调节Na泵介导的ATP/ADP交换和Na/Na 与钙泵的类似模式交换。 这些模式提供 关于配体结合和释放的顺序的信息， 泵循环的Na（或Ca）部分的稳态操作。 二.表征化学改性剂和抑制剂。 尤其涉及 确定伊红抑制位点、FITC位点之间的关系 和ATP位点。 三.表征自抑制肽相互作用。 尤其涉及 确定相互作用的一些静电和空间限制 在C-末端自抑制肽C20 W和 蛋白水解激活的钙泵。 这3种互补的方法将为评估提供基础 未来的化学和基因操作。 比如说 Ca/Ca交换中涉及的构象可能降低反应性或 与FITC反应的赖氨酸的可及性。 实验方案是检测配体，抑制剂 和肽对由人红细胞制成的由内而外的囊泡中钙摄取的影响 血细胞和检查钙泵的其他操作模式。 钙的瞬时升高对于以下过程中的信号传导很重要， 肌肉收缩、细胞分化和细胞增殖。 的Ca 泵将Ca恢复到基础水平，因此有助于终止 信号了 钙泵活性的缺陷会导致钙的增加， 关键细胞质功能的后续改变。 这样的离子 这些改变与几种疾病状态有关， 肾和心脏衰竭、高血压和肌肉萎缩症。 镰状细胞中红细胞钙泵的改变已被报道 疾病