REGULATION OF NA+/H+ EXCHANGER IN AMPHIUMA ERYTHROCYTE

AMPHIUMA 红细胞中 NA /H 交换剂的调控

• 批准号: 6395887
• 负责人: HECTOR M MALDONADO
• 金额: $ 13.96万
• 依托单位: UNIVERSIDAD CENTRAL DEL CARIBE
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-01-01 至 2000-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6395887
• 关键词: Urodela; alternatives to animals in research; biological signal transduction; biological transport; calcium flux; calmodulin; cell biology; cell morphology; confocal scanning microscopy; enzyme activity; erythrocytes; gel electrophoresis; hydrogen ions; immunoprecipitation; membrane channels; membrane transport proteins; phosphorylation; protein isoforms; protein kinase C; protein tyrosine kinase; protein tyrosine phosphatase; sodium ion; western blottings

Na+/H+ exchanger is a broadly distributed transporter that plays a central role in a variety of cellular processes including cell volume and intracellular pH regulation. The same trasporter has also been implicated in a variety of pathological conditions including hypertension, diabetes, and the response of several tissues to hypoxic/ischemic insults. The combination of several isoforms of the transporter and their sensitivity to a variety of controlling mechanisms, have been frequently invoke as an explanation for the versatility of the exchanger and its involvement in many (and sometimes contradictory) biologicalprocesses. In this application, we propose to use the Amphiuma erythrocyte as a model system to study the regulation of this inducible transporter. Evidence fromthis and other laboratoris indicated that the activity of the transporter is regulated by protein phosphorylation, suggesting a role for protein kinases and phosphatases inthis regulation. Our working hypothesis is that: "Interactions between several protein kinases and phosphatases, acting directly on the exchange protein or indirectly on a closely associated protein, ae responsible for the regulation of the volume sensitive Amphiuma erythrocyte Na+/H+ exchanger". In order to test this hypothesis we are proposing to expand our pharmacological and biochemical approach to establish the role, and cross-talk interactions, between several signaling pathways in regulating exchange activity. Specifically, the proposed study will focused on the following kinases: casein kinase-II, protein kinase-C, tyrosine kinases, mitogen activated protein kinases, and Ca++- calmodulin dependent kinase. The role and interaction of protein phosphatases will also be established, and the ability of these kinases and phosphatases in modulating the net phosphorylation of the exchanger will be determined by immunoprecipitation studies. Emphasis will be given to the cross-talk interactions between the different signaling cascades and its net effect on exchange activity. Finally, the possibility of regulation of the exchanger by recuritment-removel of transporter to/from the plasma membrane by fluid-phase endocytosis will also be explored. Identification and characterization of the vesicles and the co-migration of the exchange protein in these organelles willbe established with the use of antibodies, in combination with confocal microscopy, and in isolated vesicles followed by immuno-blot analysis. The results of these studies shold provide fundamental information concerning the mechanism that regulate Na+/H+ exchange function, and will have broad implications for cellular physiology and pathophysiology.

Na+/H+交换器是一种分布广泛的传输器，它扮演着 在包括细胞体积在内的各种细胞过程中发挥中心作用 细胞内pH调节。同样的运输车也被 牵涉到各种病理情况，包括 高血压、糖尿病和几种组织对 缺氧/缺氧性侮辱。几种异构体的结合 转运蛋白及其对多种控制的敏感性 机制，经常被用来解释 交换机的多功能性及其参与的许多(和 有时相互矛盾)生物过程。在此应用程序中， 我们建议使用两栖动物红细胞作为模型系统来研究 对这种诱导性转运蛋白的调节。来自这一点的证据和 其他实验室表明，转运体的活动是 受蛋白质磷酸化调节，提示蛋白质的作用 在这一调节中的激酶和磷酸酶。我们的工作假说 就是：“几种蛋白激酶和 直接作用于交换蛋白或间接作用于交换蛋白的磷酸酶 在一种密切相关的蛋白质上，Ae负责调节 容量敏感型两栖动物红细胞Na+/H+交换器 为了检验这一假设，我们建议扩展我们的 用药理学和生物化学的方法来确定作用，以及 中几个信号通路之间的串扰交互作用 规范交易活动。具体而言，拟议的研究将 重点研究了以下几种激酶：酪蛋白激酶-II、蛋白激酶-C、 酪氨酸激酶、丝裂原活化蛋白激酶和钙离子 钙调蛋白依赖激酶。蛋白质的作用和相互作用 磷酸酶也将被建立，这些酶的能力 蛋白水解酶和磷酸酶在调节细胞内净磷酸化中的作用 交换剂将通过免疫沉淀研究来确定。 将重点放在两种语言之间的串扰互动 不同的信号级联及其对交换活动的净影响。 最后，通过以下方式监管交易所的可能性 递归-通过以下方式移除进入/离开质膜的转运蛋白 还将探讨液体相内吞作用。身份识别和 囊泡的表征和交换的共迁移 这些细胞器中的蛋白质将通过使用 抗体，结合共聚焦显微镜，并在分离的 然后进行免疫印迹分析。这些研究的结果 研究应提供有关的基本信息 调节Na~+/H~+交换功能的机制，具有广泛的应用前景 对细胞生理学和病理生理学的影响。