REGULATION OF NA+/H+ EXCHANGER IN AMPHIUMA ERYTHROCYTE

Na+/H+交换器是一种广泛分布的转运体， 在细胞体积等多种细胞过程中发挥核心作用 和细胞内pH调节。 同样的运输机也被 涉及多种病理状况，包括 高血压、糖尿病以及几种组织对 缺氧/缺血损伤。 几种同种型的组合， 转运蛋白及其对各种控制的敏感性 机制，已被频繁调用作为一种解释， 交换器的多功能性及其在许多（和 有时是矛盾的）生物过程。 在本申请中， 我们建议使用两栖鱼红细胞作为模型系统， 这种诱导型转运蛋白的调节。 证据表明， 其他实验室指出，转运蛋白的活性是 受蛋白质磷酸化调节，表明蛋白质 激酶和磷酸酶在此调节中的作用。 我们的工作假设 是：“几种蛋白激酶之间的相互作用， 磷酸酶，直接或间接作用于交换蛋白 一种密切相关的蛋白质，负责调节 体积敏感型两栖鱼红细胞Na+/H+交换器。 在 为了验证这一假设，我们建议扩大我们的 药理学和生物化学方法来确定作用，以及 在几个信号通路之间的串扰相互作用， 规范交易所活动。 具体而言，拟议研究将 集中于以下激酶：酪蛋白激酶-II，蛋白激酶-C， 酪氨酸激酶、促分裂原活化蛋白激酶和Ca++- 钙调蛋白依赖性激酶 蛋白质的作用和相互作用 也将建立磷酸酶，这些磷酸酶的能力 激酶和磷酸酶在调节蛋白质的净磷酸化中的作用 将通过免疫沉淀研究来确定交换剂。 重点将放在串扰之间的相互作用， 不同的信号级联及其对交换活动的净效应。 最后，通过以下方式调节交换器的可能性： 再循环-转运蛋白被清除到质膜/从质膜上清除 还将探索液相内吞作用。 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 囊泡，然后进行免疫印迹分析。 的结果予以 研究应提供有关 调节Na+/H+交换功能的机制，并将具有广泛的 对细胞生理学和病理生理学的影响。

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.