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：
7539561
负责人：
Zijian Xie
金额：
$ 4.25万
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/7539561
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 pressure 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

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.

Na/k-ATPase属于P型ATPases的家族，被发现为能量转导离子泵。 Na/K-ATPase和其他P型ATPases之间的主要区别在于它的结合能力心源类固醇（CI），例如ouabain。内源性CTS通过其调节血压对脉管系统和肾脏盐处理的影响，植物衍生的CT被用作药物多于 200年。最近，我们证明了Na/k-ATPase是一个重要的受体，它位于 Caveolae并与非受体酪氨酸激酶SRC直接相互作用。我们现在知道低的影响乌巴因在许多细胞功能上的剂量并不是由于对ATPase的简单抑制作用。而是他们需要激活Na/K-ATPase/SRC受体复合物。此外，Na/k-atpase包含多个结合基序（域），能够将SRC和其他信号传导酶带到其效应子，例如离子通道。这些发现导致研究领域看着NA/K-ATPase而不是仅作为离子泵，但还是经典的受体复合物。范式的这种转变带来了重要问题：此信号NA/K-ATPase在调节中有哪些调节目的与内源性CT的生理学相关的细胞功能（例如，血压控制）？这提出了通过研究该受体复合物的最可能目标来弥合这一差距的应用，即 CA2+信号模块，因为已知细胞内Ca2+的变化在调节中起关键作用血管功能和肾脏盐处理。具体而言，我们将研究Na/K-ATPase如何整合在肾上皮细胞中的功能性CA2+信号模块中多个成分。我们建议使用合并生化，细胞，遗传和动态成像方法的结合1）定义分子 Na/K-ATPase将SRC/PLC-Y/PKC和IPS受体整合到动态Ca2+的机制中信号模块； 2）揭示Na/K-ATPase与IP之间的相互作用的破坏受体会影响IPS受体运输和Ouabain诱导的Ca2+信号传导； 3）识别等离子体与Na/k-ATPase相互作用并负责Ouabain诱导的Ca2+的膜通道涌入。这些研究不仅将将Na/K-ATPase的新发现的受体功能与肾脏相关联 CTS的生理学，但还提供了有关形成Ca2+信号的详细机械信息最终将为我们提供新目标的模块，以开发肾脏的治疗方法涉及细胞内CA调节功能障碍的心血管疾病。钙是通用的第二使者，在控制肾脏和心血管中起着至关重要的作用功能。细胞内钙调节异常将导致肾脏和心脏病，例如高血压。我们正在使用一个简单的模型来剖析非常重要的钙控制的形成肾细胞中的系统，并研究我们如何操纵该系统以最终开发新的预防肾脏和心血管疾病的方法。