Cellular/molecular Na,K-ATPase regulation in choroid plexus

描述（由申请人提供）：一个长期目标是了解钠泵（Na， k - atp酶）调节的生化和细胞机制，钠泵是脑脊液（CSF）分泌的驱动力。另一个是了解这种调节是如何与分泌上皮中独特的形态变化协调的。我们有数据表明，顶端膜的磷酸化事件协调了Na， k - atp酶的调节和分泌细胞形态的调节。脑脊液由脑室的脉络膜丛产生，并通过其他结构流出。临床上当流出通道被阻断时，会导致脑积水（婴儿）或颅内高压（成人）。我们将探讨降低脉络膜丛Na、k - atp酶活性的内源性机制，包括口渴、抗利尿激素、血清素和碳酒精。拟议的研究有三个互补的重点。第一个目的是使用生物化学，原代培养和细胞遗传操作的工具来验证脉络膜丛Na， k - atp酶是由激酶介导的辅助蛋白磷酸化调节的，磷酸化蛋白（FXYD1），并改变酶的动力学。比较野生型和磷蛋白敲除小鼠的培养物可以分离直接和间接影响。第二个目的是使用显微镜和生物化学工具来研究这种调节是否需要从微绒毛到根尖下隔室的再分配。第三个目的是使用野生型、敲除磷酸化蛋白和脑积水小鼠来验证磷酸化蛋白是调节Na、k - atp酶和脉络膜细胞激活状态的必要环节的假设。目的是验证以下几个假设：1)通过大分子复合物，Na， k - atp酶和顶端刷状边缘的形态变化有协调的调节；2)敲除磷蛋白小鼠对Na、k - atp酶抑制和脉络膜细胞形态改变的生理信号反应存在缺陷；3)发生脑积水的小鼠品系表现出磷酸化增强，细胞形态变化增加，Na， k - atp酶降低。相关性：需要更好地了解脑脊液分泌器官的这些基本机制，以促进发现减少脑脊液分泌的新方法，以取代手术分流或至少在手术前稳定脆弱的婴儿。

DESCRIPTION (provided by applicant): One long term objective is to understand the biochemical and cellular mechanisms of the regulation of the sodium pump (Na,K-ATPase), which is the driving force for cerebrospinal fluid (CSF) secretion. The other is to understand how that regulation is coordinated with the unique morphological changes in the secretory epithelium. We have data that indicate that phosphorylation events at the apical membrane coordinate the regulation of Na,K-ATPase with the regulation of secretory cell morphology. CSF is produced by the choroid plexuses in the brain's ventricles, and it flows out through other structures. Clinically when the outflow pathways are blocked, hydrocephalus (infants) or intracranial hypertension (adults) results. There are endogenous mechanisms that reduce Na,K-ATPase activity in the choroid plexus that we will investigate, including thirst, vasopressin, serotonin, and carbachol. The proposed research has three complementary thrusts. The first aim uses the tools of biochemistry, primary cultures, and genetic manipulation of cells to test the hypothesis that choroid plexus Na,K-ATPase is regulated by kinase-mediated phosphorylation of an accessory protein, phospholemman (FXYD1), with a change in the kinetics of the enzyme. Comparison of cultures from wild type and phospholemman knockout mice will allow direct and indirect effects to be separated. The second aim uses the tools of microscopy and biochemistry to investigate whether the regulation entails redistribution from microvilli to a subapical compartment. The third aim uses wild type, phospholemman knockout, and hydrocephalic mice to test the hypothesis that phospholemman is an obligatory link to regulation of both Na,K-ATPase and choroid cell activation state. The aims test several hypotheses: 1) that there is concerted regulation of Na,K-ATPase and morphological change at the apical brush border through macromolecular complexes; 2) that the phospholemman knockout mouse has defective responses to physiological signals to inhibit Na,K-ATPase and change choroid cell morphology; and 3) that a mouse strain that develops hydrocephalus shows enhanced phospholemman phosphorylation, increased cell morphology changes, and reductions in Na,K-ATPase. Relevance: A better understanding of these basic mechanisms in the CSF-secreting organ is needed to facilitate new discoveries of ways to reduce CSF secretion, either to replace surgical shunts or to at least stabilize fragile infants prior to surgery.