Physiological Significance of Na,K-pump Diversity

Na. K泵（即，Na，K-ATP酶）是强心苷类的主要药理学受体， 洋地黄和哇巴因它是一种跨质膜蛋白复合物，介导蛋白质的交换， Na+和K+以消耗代谢能量为代价。由此产生的离子梯度提供了营养物质的基础 摄取、细胞内pH和Ca ++的控制、膜兴奋性和体积调节。它遵循 钠钾泵是细胞内稳态中心。它的组件子单元以多种形式存在， 这种亚基多样性的生理相关性仍然未知。总体而言， 催化，α亚基，存在于四种异构体中，而相关的蛋白质，β亚基，存在于 三.最近的工作表明，异构体之间的主要功能区别是它们与 调节蛋白如蛋白激酶C（PKC），其也以多种形式存在。的四项具体 该项目的目的是明确评估这种蛋白质多样性在调节中所起的作用。第一个目标 将使用哺乳动物细胞中的异源表达来评估泵中的同种型特异性差异， 通过刺激PKC启动调节。第二个具体目标将决定机制 再次使用异源表达策略，揭示了同种型特异性差异。第三个目标 将确定PKC同工酶在泵同工酶调节中的作用，使用特定的 抑制剂和基因技术来抑制激酶活性。最后，第四个具体目标将建立 亚基异质性在天然表达多种形式的细胞中的调节结果， 抑制内源性同种型。综合起来，这些研究将提供关于 由蛋白质多样性在泵的调节中发挥作用。更重要的是，完成这些研究将 提高我们对活性Na+、K+亚基多样性及其生理重要性的全面认识 运输 这些研究将利用培养中已建立的细胞系。由于基本的作用， 细胞内稳态中的Na. K-泵，它很好地适用于这些模型的使用。事实上，这个项目 扩展了现有非哺乳动物模型系统的有用性，与NCRR的目标一致。

The Na.K-pump (i.e., Na,K-ATPase) is the major pharmacological receptor for cardiac glycosides such as digitalis and ouabain. It is a plasma membrane-spanning protein complex that mediates the exchange of Na+ and K+ at the expense of metabolic energy. The resulting ionic gradients provide the basis for nutrient uptake, control of intracellular pH and Ca++, membrane excitability, and volume regulation. It follows that the Na.K-pump is central to cellular homeostasis. Its component subunits exist in multiple forms, but the physiological relevance of this subunit diversity remains unknown. The primary contributor to overall catalysis, the alpha subunit, exists in four isoforms, while an associated protein, the beta subunit, exists in three. Recent work suggests that the major functional distinction among the isoforms is their interaction with regulatory proteins such as protein kinase C (PKC), which also exist in multiple forms. The four specific aims of this project will evaluate explicitly the role played by this protein diversity in regulation. The first aim will use heterologous expression in mammalian cells to evaluate the isoform-specific differences in pump regulation initiated by stimulation of PKCs. The second specific aim will determine the mechanisms underlying the isoform-specific differences, again using the heterologous expression strategy. The third aim will identify the contribution played by isozymes of PKC in the regulation of pump isoforms, using specific inhibitors and genetic techniques to suppress kinase activity. Finally, the fourth specific aim will establish the regulatory consequences of subunit heterogeneity in cells that naturally express multiple forms by suppressing the endogenous isoforms. Takentogether, these studies will provide crucial data on the role played by protein diversity in regulation of the pump. More importantly, completion of these studies will increase our overall understanding of subunit diversity and its physiological importance in active Na+,K+ transport. These studies will take advantage of established cell lines in culture. Because of the fundamental role of the Na.K-pump in cellular homeostasis, it lends itself well to the use of these models. Indeed, this project expands the usefulness of existing nonmammalian model systems,consistent with the goals of the NCRR.