NA K ATPASE ISOZYME LOCALIZATION AND FUNCTION IN CNS

描述：这是一个继续研究Na，K-ATP酶的建议 在中枢神经系统（CNS）中的表达和功能。 这种酶 控制跨质膜的离子梯度， 和细胞外液的体积。 它消耗大脑50%的能量 视网膜占70%。 由于它的几种异构体在底物上不同， 对调节的亲和力和敏感性，定义亚型的模式 表达对于理解酶在CNS中的作用是重要的。 该实验室已经开发出针对三种亚型的抗体， Na，K-ATP酶α亚基以及三种β亚基同种型，和 用于小脑和细胞培养中的定位研究。 一 第三种γ亚基的研究很少。 有人建议， 人类基因组计划将迅速导致识别所有 这种酶的亚单位亚型，以及相关的H，K-ATP酶。 在 在这种情况下，该建议的第一个目的是产生异构体特异性的 针对CNS中表达的所有亚基的抗体，以确定其 通过免疫细胞化学和共聚焦显微镜观察脑中的分布， 视网膜，培养的星形胶质细胞，视神经和分泌结构，如 脉络丛和睫状体。 将对新亚基的cDNA进行测序 和表征了 第二个目标将研究的功能特性， 哺乳动物细胞和转染子中的不同同种型组合， 回答有关其生理作用的问题，并确定 星形胶质细胞钾转运特性的分子基础。 第三个目标 使用Na，K-ATP酶活性组织化学测定来检测活性 不同的异构体在原位，并测试假设，减少 缺血后的酶活性是细胞和亚型选择性的， 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组合。

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.