Cell Structure-Function of Na pump Assembly

细胞结构-钠泵组件的功能

• 批准号: 6848900
• 负责人: CRAIG GATTO
• 金额: $ 21万
• 依托单位: ILLINOIS STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-08-01 至 2009-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6848900
• 关键词: Baculoviridae; X ray crystallography; active sites; adenosine triphosphate; binding sites; cell biology; conformation; crystallization; enzyme mechanism; enzyme structure; immunoprecipitation; membrane transport proteins; protein structure function; sodium potassium exchanging ATPase

DESCRIPTION (provided by applicant): The sodium pump has been the target for the therapeutic treatment of congestive heart failure with cardiac glycosides for more than a century. Unfortunately, there is still very little known about the mechanism of cardiac glycoside inhibition. Experimental studies over the past three decades have established some relationships between the biochemical reactions catalyzed by the sodium pump and the transport reactions it mediates. However, the molecular mechanism by which the hydrolysis of ATP is coupled to the "uphill" movement of ions remains a mystery. This probably stems from the fact that there remain key components about the structure-function relationship of this protein yet to be elucidated. For example, why are the Na,K and H,K-ATPases the only members of this protein superfamily to have an obligatory alpha- and beta-subunit? Our long term goal is to gain a complete understanding of the sodium pump transport mechanism, as well as defining the other cellular roles mediated by Na,K-ATPase. For example, the Na pump has been shown to associate with several cytosolic proteins and may serve as a plasma membrane docking site for these signaling molecules (e.g. PI-3 kinase). However, before we can attempt to resolve the basis for these hetero protein-protein interactions, we must first identify the characteristics for Na pump assembly and trafficking. Indeed, there is still considerable debate over the quaternary structure of the Na,K-ATPase, a question directly examined in this proposal (AIMs I-III). The specific experiments outlined in this proposal will exploit insect cell expression (i.e. Trichoplusia ni High Five cells), devoid of endogenous Na,K-ATPase, to address unresolved issues concerning the assembly, trafficking, and oligomeric state of the Na pump en route to the plasma membrane. Wild-type and mutant sheep alpha-subunits will be introduced into the insect cells via the baculovirus system and the pump maturation process will be followed from endoplasmic reticulum to plasma membrane by membrane fractionation and co-immunoprecipitation. The results from this work will provide important new information about the Na pump quaternary structure. The sodium pump is vital in a variety of organs for fluid and electrolyte balance. These processes are in dynamic equilibrium, and this equilibrium can become disrupted in a variety of disease states. In addition, it is becoming apparent that the Na pump contributes more to the cell than ionic homeostasis and the experiments proposed here are crucial if we hope to understand the complex cell biological role mediated by the Na pump.

描述（由申请人提供）：一个多世纪以来，钠泵一直是心脏糖苷治疗充血性心力衰竭的目标。不幸的是，关于心糖苷抑制的机制仍然知之甚少。近三十年来的实验研究已经建立了钠泵催化的生化反应与其介导的输运反应之间的一定关系。然而，ATP水解与离子“上坡”运动耦合的分子机制仍然是一个谜。这可能是因为该蛋白的结构-功能关系仍有关键成分有待阐明。例如，为什么Na K和H K- atp酶是这个蛋白质超家族中唯一具有强制性α和β亚基的成员？我们的长期目标是全面了解钠泵转运机制，以及确定由Na， k - atp酶介导的其他细胞作用。例如，钠泵已被证明与几种细胞质蛋白相关，并可能作为这些信号分子（例如PI-3激酶）的质膜对接位点。然而，在我们试图解决这些异蛋白-蛋白相互作用的基础之前，我们必须首先确定钠泵组装和运输的特征。事实上，关于Na， k - atp酶的四元结构仍然存在相当大的争论，这是本提案（AIMs I-III）中直接研究的一个问题。本提案中概述的具体实验将利用缺乏内源性Na， k - atp酶的昆虫细胞表达（即Trichoplusia ni High Five细胞）来解决有关Na泵在通往质膜途中的组装，运输和低聚状态的未解决问题。将野生型和突变型羊α -亚基通过杆状病毒系统导入昆虫细胞，通过膜分离和共免疫沉淀从内质网到质膜进行泵成熟过程。这一研究结果将为钠泵第四纪结构的研究提供重要的新信息。钠泵在各种器官中保持液体和电解质平衡是至关重要的。这些过程处于动态平衡，而这种平衡在各种疾病状态下会被破坏。此外，越来越明显的是，钠泵比离子稳态对细胞的贡献更大，如果我们希望了解钠泵介导的复杂细胞生物学作用，这里提出的实验是至关重要的。