PHYSIOLOGICAL SIGNIFICANCE OF NA,K-PUMP ALPHA ISOFORMS

描述：（申请人摘要） Na，K泵（即，Na，K-ATP酶）是主要的药理学受体， 强心苷如洋地黄和哇巴因。它是一种等离子体 介导Na+和K+交换的跨膜蛋白复合物， ATP水解产生的代谢能的消耗。关键职能 包括维持电化学梯度， Na+和K+穿过质膜，Ca++、糖和氨基的运动 酸通过共转运系统，以及盐和水的运输 上皮细胞该泵由两个不同的亚基，α和β。的 α亚基含有所需底物的结合位点， 在离子转运过程中被泵和瞬时磷酸化。它存在于 至少三种不同的亚型（α 1、α 2和α 3）， 酶动力学和对激素的反应。这些差异源于 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 结构改变引起酶动力学的深刻变化， 第二个信使。尽管如此，结构与功能的关系 这些变化背后的原因尚不清楚。位点特异性体外诱变和 DNA介导的基因转移技术将被用来探索后果 氨基末端的修饰。这将证明 该区域对同种型之间动力学差异的贡献。到 介导其动力学效应，氨基末端必须与另一个 在alpha subunit中。一个有希望的候选人是另一个地区， 亚基中心附近的同种型相异，并且这个发散区域 也将被改变以确定其对同种型特异性动力学的影响。 最后，氨基末端和异构体特异性 将检查蛋白激酶调节的区域。综上所述各项 这些研究将提供有关这些区域在功能上发挥作用的重要数据。 异构体之间的差异。更重要的是，完成这些研究 将增加我们对α亚基多样性及其意义的理解 在主动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.