MOLECULAR ANALYSIS OF NA+/K+ ATPASE

这项建议的目标是对 Na，K-ATPase异构体的表达、结构和功能 大脑。具体目标包括：1)Na，K，ATPase基因的调控 表情。我们将尝试描述DNA序列和 促进Na，K-ATPase细胞特异性激活的细胞因子 中枢神经系统的α3和β2亚单位基因。我们将首先使用基因 转移方法以识别Alpha3内的序列元素和 促进A基因高效和细胞特异性表达的Beta2基因 记者基恩。我们还将尝试使用逆转录病毒介导的基因 将报告基因结构直接导入到 生后大鼠视网膜。因为Alpha3和Beta2亚基自然是 在感光细胞中产生的，应该可以识别 Alpha3和Beta2基因中所需的调控元件 促进光感受器细胞特异性基因的表达。2)结构- 功能分析。我们将尝试识别a中的序列 钠亲和力差异较大的亚基 Hydraα亚基和大鼠α1亚基。构造和表达 Hydraα和大鼠Alpha1亚单位之间的嵌合体cDNA应该 允许识别与Na+相互作用并有助于 Na+结合。为了绘制详细的区域地形图 亚基，我们将使用表位加法来创建一组α1亚基 携带插入标签的DNA。间接免疫荧光显微镜 具有与表位标签反应的单抗的转染体 应该允许我们确定是否定位了表位标记的结构域 在牢房的内部或外部。3)功能意义 亚型多样性。表达受限的细胞类型的鉴定 α亚基和β亚基的组合使比较 不同Na，K-ATPase同工酶的生化特性首字母 兴趣将集中在基板要求的比较上 松果体α3/β2同工酶和神经元酶 由α3和β1亚基组成。这种方法应该允许我们 开始了解Na，K-ATPase的功能差异 同工酶，并确定β亚基是否对 特定的阿尔法亚基对Na+和/或K+的亲和力。

The objective of this proposal is the molecular analysis of the expression, structure, and function of Na,K-ATPase isoforms expressed in brain. Specific aims include: 1) Regulation of Na,K,ATPase gene expression. We will attempt to characterize the DNA sequences and cellular factors that promote cell-specific activation of the Na,K-ATPase alpha3 and beta2 subunit genes in the CNS. We will first use gene transfer methods to identify sequence elements within the alpha3 and beta2 genes that promote efficient and cell-specific expression of a reporter gene. We will also attempt to use retrovirus-mediated gene transfer to introduce reporter gene constructs directly into the postnatal rat retina. Because alpha3 and beta2 subunits are naturally produced in photoreceptor cells, it should be possible to identify regulatory elements within the alpha3 and beta2 genes that are required to promote photoreceptor cell-specific gene expression. 2) Structure- function analysis. We will attempt to identify sequences within the a subunit responsible for the wide variation in sodium affinity between the Hydra alpha subunit and rat alpha1 subunit. Construction and expression of chimeras between Hydra alpha and rat alpha1 subunit cDNAs should permit identification of sites that interact with Na+ and contribute to Na+ binding. In order to develop a detailed topographical map of the a subunit, we will use epitope addition to create a panel of alpha1 subunit cDNAs carrying insertional tags. Indirect immunofluorescence microscopy of transfectants with a monoclonal antibody reactive with the epitope tag should permit us to determine whether an epitope-tagged domain is located on the inside or outside of the cell. 3) Functional significance for isoform diversity. The identification of cell types expressing limited combinations of alpha and beta subunits makes it possible to compare the biochemical properties of distinct Na,K-ATPase isoenzymes. Initial interest will focus on a comparison of the substrate requirements of the pineal gland alpha3/beta2-containing isoenzyme and the neuronal enzyme composed of alpha3 and beta1 subunits. This approach should allow us to begin to understand the functional differences between Na,K-ATPase isoenzymes and determine whether the beta subunit contributes to the affinity of a specific alpha subunit for Na+ and/or K+.