NA K ATPASE ISOZYME LOCALIZATION AND FUNCTION IN CNS

描述：这是一项继续研究Na，K-ATPase的建议 中枢神经系统(CNS)的表达和功能。这种酶 控制跨质膜的离子梯度以及组成 和细胞外液的体积。它消耗了50%的大脑能量 70%在视网膜中。因为它的几种异构体在底物上不同 亲和力和对调节的敏感性，定义异构体的模式 表达对于了解该酶在中枢神经系统中的作用很重要。 这个实验室已经开发出针对三种亚型的抗体。 Na，K-ATPaseα亚基以及三种β亚基亚基的亚基，以及 用于小脑和细胞培养的定位研究。一个 对第三个伽马亚基的研究很少。有人建议说，进展 关于人类基因组计划将迅速导致ALL的识别 该酶的亚基异构体，以及相关的H，K-ATPase。在……里面 在这种情况下，这项提议的第一个目标是产生特定于同种异构体的 针对中枢神经系统中表达的所有亚单位的抗体，以确定其 免疫细胞化学和共聚焦显微镜在脑内的分布 视网膜、培养的星形胶质细胞、视神经和分泌结构 脉络丛和睫状体。新亚基的cDNA将被测序 并以此为特征。第二个目标将研究的功能性质 哺乳动物细胞和转染体中不同的亚型组合 回答有关他们的生理角色的问题，并确定 星形胶质细胞钾转运特性的分子基础。第三个目标 是使用Na，K-ATPase活性的组织化学方法来检测活性 不同的异构体在原位，并检验假设减少 缺血后的酶活性对细胞和异构体具有选择性和重要性 作为一种神经保护机制。这些目标将通过使用 细胞、分子和免疫学方法的结合。

DESCRIPTION: This is a proposal to continue studies of Na,K-ATPase expression and function in the central nervous system (CNS). This enzyme controls ion gradients across plasma membranes, as well as the composition and volume of extracellular fluid. It consumes 50% of energy used in brain and 70% in retina. Since its several isoforms differ in substrate affinities and susceptibility to regulation, defining patterns of isoform expression is important to understanding roles of the enzyme in the CNS. This laboratory has developed antibodies specific for three isoforms of the Na,K-ATPase alpha subunit as well as for three beta subunit isoforms, and used these for localization studies in cerebellum and in cell culture. A third gamma subunit has been little studied. It is suggested that progress on the Human Genome Project will rapidly lead to identification of all subunit isoforms of this enzyme, as well as the related H,K-ATPases. In this context, the first aim of this proposal is to produce isoform-specific antibodies to all of the subunits expressed in the CNS, to determine their distributions by immunocytochemistry and confocal microscopy in brain, retina, cultured astrocytes, optic nerve and secretory structures such as choroid plexus and ciliary body. cDNAs for new subunits will be sequenced and characterized. The second aim will investigate functional properties of different isoform combinations in mammalian cells and transfectants to answer questions about their physiological roles and to determine the molecular basis for astrocyte potassium transport properties. The third aim is to use a histochemical assay for Na,K-ATPase activity to detect activity of different isoforms in situ, and to test the hypothesis that reduced enzyme activity after ischemia is cell- and isoform-selective and important as a neuroprotective mechanism. These aims will be accomplished using a combination of cellular, molecular and immunological methodologies.