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+对它们的电化学梯度。 它是最大的 中枢神经系统的能量消耗者，占40-50%， ATP水解。 它有三种遗传上不同的亚型， 催化（α）亚基及其糖蛋白的两种亚型 （β）亚基。 Na，K-ATP酶的基本作用是众所周知的，但 我们建议确定它的调节和参与胶质细胞K+ 通过研究在不同脑细胞中表达的同种型来清除。 第一个假设是Na，K-ATP酶β亚基，像α亚基一样， 亚基，在神经系统中具有不同的细胞分布。 新 同种型特异性单克隆抗体将用于表征Na， K-ATP酶β亚基，并将其定位于已鉴定的细胞类型， 组织切片上的免疫细胞化学。 抗体将广泛用于 特性，特异性将严格确定与新的 利用M13噬菌体随机肽库的分子方法。 在我们 在神经胶质细胞的原代培养中，我们看到了前所未有的分化的 细胞类型。 混合文化和分离文化的组成， Na，K-ATP酶同工型基因表达，将被表征并与 胶质细胞表型。 我们有新的证据表明Na，K-ATP酶的α 1亚型 在原代培养的神经胶质细胞中， 第二个假设是，酶被改变，以更好地发挥K+的功能， 泵 膜结合Na，K-ATP酶的生化特性将 并研究完整胶质细胞中Na，K-ATP酶的活性 通过离子转运和组织化学。 Na，K-ATP酶的性质 不同的神经胶质类型将被单独表征。 我们将 通过以下步骤确定改变的功能是否涉及α 2、β 1和β 2 克隆亚基的表达。 一个令人兴奋的发展是， 无效突变小鼠在断奶前死亡，伴有CNS星形胶质细胞肿胀 endfeet，表明β 2对神经胶质功能具有独特的重要性。 我们将研究神经胶质细胞的存活和ATP酶的特性， 从这只转基因小鼠身上。 现有证据表明，同种型对Na+的亲和力不同， ATP和强心苷，以及对 未知的细胞内因子。 第三个假设是， 同种型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-ATP酶的生理状态（静息或 翻转）。 这将通过生物化学进行严格评估 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.