CARDIAC NA,K-ATPASE GENES: STRUCTURE & FUNCTION

拟议项目的目标是隔离、表征和 使用编码Na，K-的DNA序列作为分析工具 大鼠心脏ATPaseα和β亚基。通过比较速率 具有先前特征的α和β的心脏cDNA 我们将从大鼠脑中提取亚单位基因，以确定Na、K- 不同初级序列的ATPase分子存在于 心脏组织。Na，K-的cDNA探针的可用性 ATPase将允许我们解决有关以下方面的基本问题 影响心肌Na，K-ATPase的调控机制。 这些研究包括：a)特定组织中ATPase基因表达的分析 心脏的细胞。我们将使用原位杂交技术来 确定ATPase mRNA的替代形式是否 在发育中和成年大鼠心脏的特定细胞中表达。 B)ATPase基因表达的调控。通过核酸 杂交技术，我们将确定生理上的 应激可导致Na，K-ATPase活性的调节 通过改变细胞内Na，K-ATPase基因的表达。 C)ATPase基因家族的组织。为了 了解Na，K-ATPase亚型的分子基础 多样性，我们将尝试确定 大鼠的ATPase基因，并检测它们的组织 基因。这一方法还可能导致识别 可能控制细胞和组织特异性的调节区 Na，K-ATPase基因家族的表达。D)发展 Na，K-ATPase抗体。初选地点的确定 ATPase亚型之间的序列差异将构成基础 用于开发一组抗体，能够 识别Na，K-ATPase的特定亚型。这些 抗体将被用来洞察解剖位置 ATPase亚型及其机制的研究进展 生物发生和亚基组装。编码基因的介绍 将ATPase的阿尔法亚单位转化为哺乳动物细胞 允许我们研究两国之间关系的许多方面 Na，K-ATPase的结构和功能。例如, 哇巴因-核糖核酸嵌合分子的构建 耐药和哇巴因敏感形式的ATPase应允许 美国将定义ATPaseα亚基负责的区域 用于区分哇巴因敏感度。这种方法， 再加上定点突变，应该会使它成为可行的 鉴定和研究Na，K-ATPase中的其他功能结构域。

The goal of the proposed project is to isolate, characterize and use as an analytical tool the DNA sequences coding for the Na,K- ATPase alpha and beta subunits from rat heart. By comparing rat heart cDNAs with the previously characterized alpha and beta subunits genes from rat brain we will determine whether Na,K- ATPase molecules of differing primary sequence are present in heart tissue. The availability of cDNA probes for the Na,K- ATPase will permit us to address basic issues with respect to control mechanisms which affect myocardial Na,K-ATPase. These include: a) Analysis of ATPase gene expression in specific cells of the heart. We will use in situ hybridization techniques to determine whether alternative forms of ATPase mRNA are expressed in specific cells of the developing and adult rat heart. b) Regulation of ATPase gene expression. By nucleic acid hybridization techniques, we will determine whether physiological stress can lead to regulation of the activity of the Na,K-ATPase in a cell via alteration in the expression of Na,K-ATPase mRNA. c) Organization of the ATPase Gene Family. In order to understand the molecular basis for Na,K-ATPase isoform diversity, we will attempt to determine the number of copies of the ATPase gene in rat and examine the organization of these genes. This approach could also lead to the identification of regulatory regions which may control cell and tissue-specific expression of the Na,K-ATPase gene family. d) Development of Na,K-ATPase antibodies. Identification of sites of primary sequence difference between ATPase isoforms will form the basis for the development of a panel of antibodies capable of recognizing specific isoforms of the Na,K-ATPase. These antibodies will be used to gain insight into the anatomical location of ATPase isoforms and the mechanisms underlying ATPase biogenesis and subunit assembly. Introduction of the genes coding for the alpha subunit of the ATPase into a mammalian cell will allow us to study many aspects of the relationship between the structure and function of the Na,K-ATPase. For example, construction of chimeric cDNA molecules between ouabain- resistant and ouabain sensitive forms of the ATPase should permit us to define the region of the ATPase alpha subunit responsible for differential ouabain sensitivity. This type of approach, coupled with site specific mutagenesis, should make it feasible to identify and study other functional domains in the Na,K-ATPase.