MOLECULAR ANALYSIS OF NA+/K+ ATPASE

本提案的目的是对 表达，结构和功能的Na，K-ATP酶异构体表达， 个脑袋 具体目标包括：1）Na，K，ATP酶基因的调控 表情 我们将尝试描述DNA序列， 促进Na，K-ATP酶细胞特异性激活的细胞因子 α 3和β 2亚基基因。 我们将首先使用基因 转移方法以鉴定α 3内的序列元件， β 2基因，促进有效的和细胞特异性的表达， 报告基因 我们还将尝试使用逆转录病毒介导的基因 转移以将报告基因构建体直接引入到 出生后大鼠视网膜。因为α 3和β 2亚基 在感光细胞中产生，应该可以识别 α 3和β 2基因内的调控元件， 以促进感光细胞特异性基因表达。 2)结构- 功能分析 我们将尝试识别a中的序列 亚基负责钠亲和力之间的广泛变化， 水螅α亚单位和大鼠α 1亚单位。 构建及表达 水螅α和大鼠α 1亚基cDNA之间的嵌合体应该 允许识别与Na+相互作用并有助于 Na+结合。 为了绘制一幅详细的地形图， 亚基，我们将使用表位添加来创建一组α 1亚基 携带插入标签的cDNA。 间接免疫荧光显微术 具有与表位标签反应的单克隆抗体的转染子 应该允许我们确定表位标记的结构域是否位于 在细胞的内部或外部。 3)功能意义 同种型多样性 细胞类型的鉴定表达有限 α和β亚基的组合使得比较 不同的Na，K-ATP酶同工酶的生化特性。 初始 感兴趣的将集中在比较基板的要求 松果体α 3/β 2同工酶和神经元酶 由α 3和β 1亚基组成。 这种方法应该使我们能够 开始了解Na，K-ATP酶的功能差异 同工酶，并确定β亚基是否有助于 特异性α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+.