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+离子对他们的电化学梯度，与水解 ATP它被认为是世界上最大的能源消费国。 中枢神经系统，占ATP水解的30 - 40%。三 不同的Na，K-ATP酶同工酶在CNS中表达，并且在一些CNS中表达。 这些实例在不同的细胞类型中具有相当显著的局限性。 它们各自的特性和生理作用尚不清楚。 现有的证据表明，同工酶可能不同， ATP和强心苷的亲和力，以及它们对 由未鉴定的细胞内因子调节。此外，缺血性 据报道，损伤会导致其中一种选择性且快速的失活 两种同工酶的结合该提案的假设是， CNS中的Na，K-ATP酶同工酶在不同的细胞类型中表达， 它们对ATP的亲和力或对调节或降解的敏感性 可能加重或减轻缺血性损伤和水肿。的 Na，K-ATP酶的性质可能在解释不同的 不同种类的神经元对缺血的脆弱性。 该提案有几个目标：确定哪些细胞类型表达 研究其功能特性，并预测 所述同工酶将在边缘灌注的组织中保持活性;以及 研究其失活继发于细胞内Ca2+升高。 我们将利用免疫细胞化学与特异性单克隆抗体， 确定Na，K-ATP酶在细胞和亚细胞中的分布， 大鼠CNS。我们将分离同工酶， 对ATP、Na和K+的亲和力，以及研究 GTP作为底物。最后，我们将探讨 通过Ca 2+依赖的蛋白水解修饰或灭活每种同工酶 钙调蛋白介导的Ca2+抑制。这项基础研究应该奠定 对离子运动、能量、 消耗，以及它们在缺血性脑中的细胞死亡和水肿中的作用， 有助于详细了解离子泵的功能。

The Na,K-ATPase is the plasma membrane enzyme that actively transports Na + and K+ ions against their electrochemical gradients, with the hydrolysis of ATP. It is thought to be the single largest consumer of energy in the central nervous system, accounting for 30-40% of ATP hydrolysis. Three different isozymes of the Na,K-ATPase are expressed in the CNS, and in some instances are rather strikingly localized within different cell types. Their individual properties and physiological roles are not yet well known. Available evidence suggests that the isozymes may differ in their affinities for ATP and cardiac glycosides, and in their susceptibility to regulation by unidentified intracellular factors. In addition, ischemic injury has been reported to cause a selective and rapid inactivation of one of two isozymes in the heart. The hypothesis of this proposal is that the Na,K-ATPase isozymes in the CNS are expressed in different cell types, where their affinity for ATP or susceptibility to regulation or degradation may either exacerbate or ameliorate ischemic injury and edema. The properties of the Na,K-ATPases may play a role in explaining the different vulnerability to ischemia of different classes of neurons. The proposal has several objectives: to determine which cell types express which isozymes; to investigate their functional properties and predict which isozymes will remain active in marginally perfused tissue; and to investigate their inactivation secondary to a rise in intracellular Ca2+. We will utilize immunocytochemistry with specific monoclonal antibodies to determine the cellular and subcellular distribution of the Na,K-ATPases in the rat CNS. We will separate the isozymes, and determine their individual affinities for ATP, Na and K+, as well as investigate the possible use of GTP as a substrate. Finally we will investigate the mechanism of modification or inactivation of each isozyme by Ca 2+-dependent proteolysis and by calnaktin-mediated Ca 2+ inhibition. This basic research should lay the groundwork for a rigorous analysis of ion movements, energy consumption, and their role in cell death and edema in ischemic brain, and contribute to a detailed understanding of ion pump function.