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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：
7457662
负责人：
Zijian Xie
金额：
$ 32.37万
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/7457662
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 Cyclophosphamide/Fluorouracil/Prednisone Diglycerides Dimethyl Suberimidate Disruption 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 Purpose 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.

说明（由申请人提供）：Na/K-ATP酶属于P型ATP酶家族，被发现为能量转换离子泵。Na/K-ATP酶与其他P型ATP酶之间的主要区别在于其结合强心类固醇（CIS）如哇巴因的能力。虽然内源性CTS通过其对血管系统和肾盐处理的影响来调节血压，但植物来源的CTS已被用作药物超过200年。最近，我们已经证明，Na/K-ATP酶是一个重要的受体，驻留在小窝和直接相互作用的Src，一个非受体酪氨酸激酶。我们现在知道，低剂量的哇巴因对许多细胞功能的影响不是由于ATP酶的简单抑制，而是需要Na/K-ATP酶/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），并能够将Src和其他信号传导酶带到其效应器，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，而且还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）中起到何种调节作用？本申请提出通过研究这种受体复合物的最可能的靶点，即Ca 2+信号传导模块来弥合这一差距，因为已知细胞内Ca 2+的变化在调节血管功能和肾盐处理中起关键作用。具体来说，我们将研究如何Na/K-ATP酶整合成一个功能性的钙离子信号模块在肾上皮细胞的多种成分。本研究拟采用生物化学、细胞学、遗传学和动态影像学相结合的方法，探讨Na/K-ATP酶整合Src/PLC-γ/PKC和IPS受体为动态Ca ~（2+）信号传导模块的分子机制，揭示Na/K-ATP酶与IPS受体相互作用的中断是否影响IPS受体的转运和哇巴因诱导的Ca ~（2+）信号传导;鉴定与Na/K-ATP酶相互作用并负责哇巴因诱导的Ca 2+内流的质膜通道。这些研究不仅将新发现的Na/K-ATP酶的受体功能与CTS的肾脏生理学联系起来，而且还提供了关于形成Ca 2+信号传导模块的详细机制信息，这将最终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中起着重要作用。细胞内钙调节的异常会导致肾脏和心脏疾病，如高血压。我们正在使用一个简单的模型来剖析肾细胞中一个非常重要的钙控制系统的形成，并研究我们如何操纵这个系统，最终开发出预防肾脏和心血管疾病的新方法。