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

Na/K-ATP酶属于P-型ATP酶家族，被发现是一种能量传递离子 泵Na/K-ATP酶与其他P型ATP酶之间的主要区别在于其结合能力 强心类固醇（CIS），如哇巴因。而内源性CTS则通过其 由于对血管系统和肾盐处理的影响，植物来源的CTS已被用作药物超过 两百年了最近，我们已经证明Na/K-ATP酶是一种重要的受体， Caveolae，并直接与Src（一种非受体酪氨酸激酶）相互作用。我们现在知道， 哇巴因对许多细胞功能的剂量不是由于ATP酶的简单抑制，而是由于它们 需要激活Na/K-ATP酶/Src受体复合物。此外，Na/K-ATPase还含有 多个结合基序（结构域），并能够将Src和其他信号传导酶带到它们的 效应器，如离子通道。这些发现使研究领域将目光投向Na/K-ATPase， 不仅作为离子泵，而且作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）？这 通过研究这种受体复合物最可能的靶点，即 Ca2+信号模块，因为细胞内Ca2+的变化已知在调节 血管功能和肾盐处理。具体来说，我们将研究Na/K-ATP酶如何整合 在肾上皮细胞中将多种成分转化为功能性Ca2+信号传导模块。我们建议使用 组合的生物化学、细胞、遗传和动态成像方法，以1）确定分子 Na/K-ATP酶将Src/PLC-γ/PKC和IPS受体整合成动态Ca 2+的机制 2）揭示Na/K-ATP酶与IPS之间的相互作用是否被破坏 受体影响IPS受体的运输和哇巴因诱导的Ca~（2+）信号传导; 与Na/K-ATP酶相互作用并负责哇巴因诱导的Ca2+的膜通道 流入。这些研究不仅将新发现的Na/K-ATP酶的受体功能与肾功能联系起来， 生理学的CTS，而且还提供了详细的机制信息的形成Ca2+信号 模块，最终将为我们提供一个新的目标，为开发治疗方法，以肾脏和 涉及细胞内Ca调节功能障碍的心血管疾病。 钙是一种普遍存在的第二信使，在肾脏和心血管系统的控制中起着重要作用。 功能细胞内钙调节的异常将导致肾脏和心脏疾病， 高血压我们用一个简单的模型来剖析一个非常重要的钙控制的形成 研究如何操纵这个系统，最终开发出新的 预防肾脏和心血管疾病的方法。

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.