PHYSIOLOGICAL SIGNIFICANCE OF NA,K-PUMP ALPHA ISOFORMS

描述：(申请人摘要) Na，K-泵(即Na，K-ATPase)是主要的药物受体 强心苷，如洋地黄和哇巴因。这是一种等离子体 膜跨膜蛋白复合体介导钠钾离子交换的研究 三磷酸腺苷水解所产生的新陈代谢能量的消耗。关键功能 由泵提供的服务包括维护电化学梯度 跨质膜的钠和钾，钙、糖和氨基的移动 酸通过共传输系统，以及盐和水的传输 上皮细胞。泵由两个不同的亚基组成，即α和β。这个 Alpha亚基包含底物所需的结合位点 泵，并在离子传输过程中被瞬时磷酸化。它存在于 至少三种不同的亚型(Alpha1、Alpha2和Alpha3)，在 酶动力学和对激素的反应。这些差异一定源于 结构多样性，但α亚型的一级结构是 几乎一模一样。这种相似性的一个例外是氨基末端，其中 结构变化会导致酶动力学和 第二信使的监管。然而，结构-功能关系 这些变化背后的原因尚不清楚。体外定点突变和 将使用DNA介导的基因转移技术来探索其后果 所有三种异构体的氨基末端修饰。这将演示 这一区域对不同亚型之间的动力学差异的贡献。至 调节其动力学效应，氨基末端必须与另一个相互作用 阿尔法亚单位内的区域。有希望的候选人是另一个领域的 亚基中心附近的异构体不同，以及这个分歧区 也将改变以确定其对异构体特定动力学的影响。 最后，氨基末端和异构体特异性改变的后果 本课程将研究蛋白激酶的调控区域。这些加在一起， 研究将提供关于这些区域在功能上所起作用的关键数据 不同亚型之间的差异。更重要的是，这些研究的完成 将增加我们对阿尔法亚基多样性及其意义的理解 在活跃的Na、K转运中。

DESCRIPTION: (Applicant's Abstract) 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 derived from ATP hydrolysis. Critical functions served by the pump include the maintenance of the electrochemical gradients for Na+ and K+ across the plasma membrane, the movement of Ca++, sugars, and amino acids via cotransport systems, and the transport of salts and water across epithelia. The pump consists of two dissimilar subunits, alpha and beta. The alpha subunit contains the binding sites for the substrates required by the pump and is phosphorylated transiently during ion transport. It exists in at least three distinct isoforms (alpha1, alpha2, and alpha3) with differences in enzyme kinetics and response to hormones. These differences must originate from structural diversity, yet the primary structures of the alpha isoforms are nearly identical. One exception to this similarity is the amino terminus, where structural alterations produce profound changes in enzyme kinetics and regulation by second messengers. Nevertheless, the structure-function relations underlying these changes are not known. Site-specific in vitro mutagenesis and DNA-mediated gene transfer techniques will be used to explore the consequences of amino terminal modification in all three isoforms. This will demonstrate the contributions of this region to differences in kinetics among the isoforms. To mediate its kinetic effects, the amino terminus must interact with another region within the alpha subunit. A promising candidate is another region of isoform dissimilarity near the center of the subunit, and this divergent region will also be altered to determine its effects on isoform-specific kinetics. Finally, the consequences of the changes in amino terminus and isoform-specific region on regulation by protein kinases will be examined. Taken together, these studies will provide crucial data on the role these regions play in functional differences among the isoforms. More importantly, completion of these studies will increase our understanding of alpha subunit diversity and its significance in active Na+,K+ transport.