NA+/K+ ATPASE ISOZYME LOCALIZATION AND FUNCTION IN CNS

Na，K-ATPase是一种主动转运Na+的质膜酶 和K+对其电化学梯度的影响。它是世界上最大的 中枢神经系统的能量消耗，占40-50% 三磷酸腺苷水解。在基因上有三种截然不同的ITS亚型 催化(α)亚单位及其糖蛋白的两种异构体 (β)亚基。Na，K-ATPase的基本作用众所周知，但 我们建议确定它对神经胶质细胞K+的调节和参与 通过研究不同脑细胞中表达的异构体来清除。 第一个假设是Na，K-ATPaseβ亚基，如阿尔法 亚单位，在神经系统中有不同的细胞分布。新的 亚型特异性的单抗将被用来表征Na， K-ATPaseβ亚基，并通过以下方式将它们定位到已识别的细胞类型 组织切片上的免疫细胞化学。抗体将广泛应用于 特性和特异性将通过一个新的 利用M13噬菌体随机多肽文库的分子方法。在我们的 神经胶质细胞的原代培养，我们看到了前所未有的分化数量 单元类型。混合和分离文化的组成以及它们的 Na，K-ATPase异构体基因的表达，将表征和相关 体内神经胶质细胞表型。 我们有新的证据表明，Na，K-ATPase的α1亚型 胶质细胞原代培养中的异常功能特征，以及 第二个假设是，这种酶被改变，以更好地发挥K+的作用 打气筒。膜结合的Na，K-ATPase的生化性质 将对完整胶质细胞的Na，K-ATPase活性进行研究 通过离子转运和组织化学。Na，K-ATPase性质的研究 不同的神经胶质类型将被单独描述。我们会 通过以下方法确定改变的函数是否涉及Alpha2、Beta1和Beta2 克隆的亚基的表达。一个令人兴奋的发展是，Beta2 零突变小鼠断奶前死亡，中枢神经系统星形胶质细胞肿胀 这表明，Beta2对神经胶质细胞的功能具有独特的重要性。 我们将在准备好的培养物中研究神经胶质细胞的存活和ATPase特性 来自这只转基因小鼠。 现有证据表明，不同亚型对Na+的亲和力不同， 三磷酸腺苷和心脏糖苷，以及对 未知的细胞内因素。第三个假设是 异构体对蛋白激酶的调节具有不同的敏感性。 A和C。我们的新结果预测PKA和PKC的作用将是 受Na，K-ATPase生理状态(静息或 翻身)。这将通过生物化学进行严格的评估 技术，使用纯化的酶和培养的细胞。

The Na, K-ATPase is the plasma membrane enzyme that actively transports Na+ and K+ against their electrochemical gradients. It is the single largest consumer of energy in the central nervous system, accounting for 40-50% of ATP hydrolysis. There are three genetically distinct isoforms of its catalytic (alpha) subunit, as well as two isoforms of its glycoprotein (beta) subunit. The fundamental role of the Na,K-ATPase is well-known, but we propose to ascertain its regulation and its participation in glial K+ clearance by investigating the isoforms expressed in different brain cells. The first hypothesis is that Na,K-ATPase beta subunits, like alpha subunits, have different cellular distributions in the nervous system. New isoform-specific monoclonal antibodies will be used to characterize the Na, K-ATPase beta subunits and localize them to identified cell types by immunocytochemistry on tissue sections. Antibodies will be extensively characterized, and specificity will be rigorously determined with a new molecular approach utilizing an M13 phage random peptide library. In our primary cultures of glia, we see an unprecedented number of differentiated cell types. The composition of mixed and separated cultures, and their Na,K-ATPase isoform gene expression, will be characterized and related to glial phenotype in vivo. We have new evidence suggesting that the alpha1 isoform of Na, K-ATPase has unusual functional characteristics in primary cultures of glia, and the second hypothesis is that the enzyme is altered to function better as a K+ pump. The biochemical properties of the membrane-bound na, K-ATPase will be determined, and Na,K-ATPase activity in intact glia will be investigated by ion transport and histochemistry. The Na,K-ATPase properties of different glial types will be characterized individually. We will determine whether the altered function involves alpha2, beta1, and beta2 by expression of cloned subunits. An exciting development is that a beta2 null mutant mouse dies before weaning with gross swelling of CNS astrocyte endfeet, demonstrating that beta2 is uniquely important for glial function. We will study glial survival and ATPase properties in cultures prepared from this transgenic mouse. Available evidence suggests that the isoforms differ in affinities for Na+, ATP and cardiac glycosides, and in susceptibility to regulation by unidentified intracellular factors. The third hypothesis is that the isoforms have different susceptibilities to regulation by protein kinases A and C. Our novel results predict that PKA and PKC actions will be affected oppositely by the physiological state of Na,K-ATPase (resting or turning-over). This will be critically evaluated with biochemical techniques, using purified enzyme and cultured cells.