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Na,K-ATPase as an Integrator of the Calcium-signaling Machinery

Na,K-ATP 酶作为钙信号传导机制的整合者

基本信息

批准号：
7653650
负责人：
Zijian Xie
金额：
$ 29.5万
依托单位：
UNIVERSITY OF TOLEDO HEALTH SCI CAMPUS
依托单位国家：
美国
项目类别：
财政年份：
2007
资助国家：
美国
起止时间：
2007-07-01 至 2011-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7653650
关键词：
1,2-diacylglycerol 1-Phosphatidylinositol 3-Kinase ATP phosphohydrolase Abbreviations Affect Binding Biochemical Biological Assay Blood Pressure Blood Vessels Calcium Calcium Signaling Cardiac Glycosides Cardiovascular Diseases Cardiovascular Physiology Caveolae Cell membrane Cell physiology Complex Cyan Fluorescent Protein Cyclodextrins Diglycerides Dimethyl Suberimidate Dithiothreitol Dominant-Negative Mutation Dose Endocytosis Enzymes Epidermal Growth Factor Receptor Epithelial Cells Family Fluorescence Resonance Energy Transfer Functional disorder Genetic Green Fluorescent Proteins Heart Diseases Hypertension ITPR1 gene Image Image Analysis In Vitro Inositol Ion Channel Ion Pumps Kidney Knock-in Mouse LLC-PK1 Cells Laboratories Lead Modeling Molecular Na(+)-K(+)-Exchanging ATPase Ouabain PH Domain Pharmaceutical Preparations Phosphatidylinositol 4,5-Diphosphate Phosphatidylinositols Phospholipase C Phosphotransferases Physiology Plants Play Propionates Protein Kinase Protein Kinase C Protein Tyrosine Kinase Proteins RNA Interference Reactive Oxygen Species Receptor Protein-Tyrosine Kinases Recruitment Activity Regulation Renal function Reporter Research Research Personnel Role Sarcoplasmic Reticulum Second Messenger Systems Signal Transduction Small Interfering RNA Sodium Chloride System Testing Therapeutic Work base blood pressure regulation crosslink extracellular kidney cell knock-down novel strategies prevent programs protein protein interaction receptor receptor function receptors for activated C kinase red fluorescent protein response second messenger time use trafficking tripolyphosphate

项目摘要

DESCRIPTION (provided by applicant): Na/K-ATPase belongs to the family of P-type ATPases and was discovered as an energy transducing ion pump. A major difference between the Na/K-ATPase and other P-type ATPases is its ability to bind cardiotonic steroids (CIS) such as ouabain. While endogenous CTS regulate blood pressure via their effects on vasculature and renal salt handling, the plant-derived CTS have been used as drugs for more than 200 years. Recently, we have demonstrated that the Na/K-ATPase is an important receptor that resides in caveolae and interacts directly with Src, a non-receptor tyrosine kinase. We know now that the effects of low doses of ouabain on many cellular functions are not due to the simple inhibition of the ATPase; rather they require the activation of the Na/K-ATPase/Src receptor complex. In addition, the Na/K-ATPase contains multiple binding motifs (domains) and is capable of bringing Src and other signaling enzymes to their effectors such as ion channels. These findings have led the research field to look at the Na/K-ATPase not only as an ion pump, but also a classical receptor complex. This shift of the paradigm has brought about an important question: which regulatory purposes does this signaling Na/K-ATPase serve in regulation of cellular functions that are relevant to the physiology of endogenous CTS (e.g. blood pressure control)? This application is proposed to bridge this gap by studying the most likely target of this receptor complex, namely the Ca2+-signaling module because changes in intracellular Ca2+ are known to play a key role in regulation of vascular function and renal salt handling. Specifically, we will investigate how the Na/K-ATPase integrates multiple constituents into a functional Ca2+-signaling module in renal epithelial cells. We propose to use a combined biochemical, cellular, genetic and dynamic imaging approach to 1) define the molecular mechanism by which the Na/K-ATPase integrates Src/PLC-y/PKC and IPS receptor into a dynamic Ca2+ signaling module; 2) reveal whether disruption of the interaction between the Na/K-ATPase and IPS receptors affects IPS receptor trafficking and ouabain-induced Ca2+ signaling; And 3) identify the plasma membrane channel (s) that interacts with the Na/K-ATPase and is responsible for ouabain-induced Ca2+ influx. These studies will not only relate the newly discovered receptor function of the Na/K-ATPase to renal physiology of CTS, but also provide detailed mechanistic information on the formation of a Ca2+-signaling module that will eventually give us a new target for developing therapeutic approaches to renal and cardiovascular diseases involving dysfunction of intracellular Ca regulation. Calcium is a universal second messenger that plays an essential role in control of kidney and cardiovascular function. Abnormality in intracellular calcium regulation will lead to both kidney and heart diseases such as hypertension. We are using a simple model to dissect the formation of a very important calcium controlling system in kidney cells and to investigate how we can manipulate this system to eventually develop new approaches to prevent renal and cardiovascular diseases.

描述（由申请人提供）：Na/K-ATP酶属于P型ATP酶家族，被发现是一种能量转导离子泵。 Na/K-ATP 酶与其他 P 型 ATP 酶之间的主要区别是其结合强心类固醇 (CIS)（例如哇巴因）的能力。虽然内源性 CTS 通过影响脉管系统和肾盐处理来调节血压，但植物源性 CTS 已被用作药物已有 200 多年的历史。最近，我们证明 Na/K-ATP 酶是一种重要的受体，存在于小凹中，并与非受体酪氨酸激酶 Src 直接相互作用。我们现在知道，低剂量哇巴因对许多细胞功能的影响并不是由于简单地抑制 ATP 酶所致；而是由于其对 ATP 酶的抑制。相反，它们需要 Na/K-ATPase/Src 受体复合物的激活。此外，Na/K-ATP酶含有多个结合基序（结构域），能够将 Src 和其他信号酶带到其效应器（例如离子通道）。这些发现使得研究领域不仅将 Na/K-ATP 酶视为离子泵，而且将其视为经典的受体复合物。这种范式的转变带来了一个重要的问题：这种信号传导 Na/K-ATP 酶在调节与内源性 CTS 生理学相关的细胞功能（例如血压控制）中起到哪些调节作用？该应用旨在通过研究该受体复合物最可能的靶点（即 Ca2+ 信号模块）来弥补这一差距，因为已知细胞内 Ca2+ 的变化在血管功能和肾盐处理的调节中发挥关键作用。具体来说，我们将研究 Na/K-ATP 酶如何将多种成分整合到肾上皮细胞中的功能性 Ca2+ 信号模块中。我们建议使用生化、细胞、遗传和动态成像相结合的方法来 1) 定义 Na/K-ATPase 将 Src/PLC-y/PKC 和 IPS 受体整合到动态 Ca2+ 信号传导模块中的分子机制； 2) 揭示 Na/K-ATPase 和 IPS 受体之间相互作用的破坏是否会影响 IPS 受体的运输和哇巴因诱导的 Ca2+ 信号传导； 3) 确定与 Na/K-ATP 酶相互作用并负责哇巴因诱导的 Ca2+ 内流的质膜通道。这些研究不仅将新发现的 Na/K-ATP 酶受体功能与 CTS 的肾脏生理学联系起来，而且还提供了有关 Ca2+ 信号模块形成的详细机制信息，最终将为我们开发涉及细胞内 Ca 调节功能障碍的肾脏和心血管疾病的治疗方法提供新的靶点。钙是一种普遍的第二信使，在控制肾脏和心血管功能方面发挥着重要作用。细胞内钙调节异常会导致肾脏和心脏疾病，例如高血压。我们正在使用一个简单的模型来剖析肾细胞中非常重要的钙控制系统的形成，并研究如何操纵该系统，最终开发出预防肾脏和心血管疾病的新方法。