Physiological Significance of Na,K-pump Diversity

描述(申请人提供)：Na，K-泵(即，Na，K-ATPase)是心脏糖苷如洋地黄和哇巴因的主要药理受体。它是一种跨越质膜的蛋白质复合体，以代谢能量为代价调节Na+和K+的交换。由此产生的离子梯度为营养吸收、细胞内pH和钙离子的控制、膜的兴奋性和体积调节提供了基础。因此，Na，K泵是细胞内稳态的中心。它的组成亚基以多种形式存在，但这种亚基多样性的生理相关性尚不清楚。整个催化反应的主要贡献者α亚基以4种亚型存在，而相关蛋白β亚基存在于3种亚基中。最近的研究表明，异构体之间的主要功能差异是它们与调节蛋白的相互作用，如蛋白激酶C(PKC)，后者也以多种形式存在。该项目的4个具体目标将明确评估这种蛋白质多样性在调控中所起的作用。第一个目标是利用哺乳动物细胞中的异源表达来评估由PKCs刺激启动的泵调节的异构体特异性差异。第二个特定目标将再次使用异源表达策略来确定异构体特异性差异的潜在机制。第三个目标将确定PKC同工酶在泵同功体调节中所起的作用，使用特定的抑制剂和基因技术来抑制激酶活性。最后，第四个具体目标将通过抑制内源性异构体，在自然表达多种形式的细胞中建立亚单位异质性的调控后果。综上所述，这些研究将提供有关蛋白质多样性在泵的调节中所起作用的关键数据。更重要的是，这些研究的完成将增加我们对亚基多样性及其在活性Na+，K+转运中的生理重要性的全面了解。这些研究将利用培养中已建立的细胞系。由于Na，K泵在细胞内稳态中的基础作用，它很适合使用这些模型。事实上，该项目扩大了现有非哺乳动物模型系统的有用性，与NCRR的目标一致。

DESCRIPTION (provided by applicant): 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 4 isoforms, while an associated protein, the beta subunit, exists in 3. 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 4 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. Taken together, 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 non-mammalian model systems, consistent with the goals of the NCRR.