Acute Regulation of the Renal Na/H Exchanger NHE-3

描述(改编自申请者的摘要)：肾脏呈 细胞外液在维持酸碱平衡中的关键作用 NaHCO3和NaCI调节血容量和血压的动态平衡 排泄物。在所有这些过程中起中心作用的一种蛋白质是Na/H 交换异构体3(NHE3)。NHE3调节绝大多数NaHCO3和NaCI 并由血流动力学和神经体液因素快速调节，以 与生物体的需求相匹配。几个实验室最近的研究表明 研究表明，NHE3可以通过数字O机制受到强烈的调控。我们有 研究表明，甲状旁腺激素(PTH)和多巴胺(DA)等激素 抑制NHE3的转运活性，而不改变其在 质膜。这种抑制与NHE3的复杂变化有关 NHE3蛋白的磷酸化和聚集性。我们假设会发生变化 NHE3磷酸化导致NHE3聚集，从而抑制 活动。在这份提案中。我们的重点是：1.绘制出氨基酸 受调控的NHE3的残留物。2.确定NHE3是否发生变化 磷酸化本身改变了它的活性。3.确定NHE3是否 聚集是NHE3磷酸化变化的结果，以及NHE3是否 集群改变了它的活动。4.确定两个NHE3结合的作用 介导NHE3聚集和磷酸化变化的蛋白质。我们会 在运输生理、生物化学、重组DNA等方面的应用 技术、免疫组织学和生物物理荧光光谱学研究 整个动物、培养细胞和纯化的蛋白质。这些研究将揭示 一种重要的上皮钠的高度基础调控机制 转运蛋白和我们对酸碱平衡障碍的进一步理解， 钠滞留和高血压。

DESCRIPTION (Adapted from the Applicant's Abstract): The kidney assumes a critical role in the maintenance of acid-base balance, extracellular fluid volume and blood pressure homeostasis via regulation of NaHCO3 and NaCI excretion. One protein that is central to all these processes is the Na/H exchanger isoform 3 (NHE3). NHE3 mediates a great majority of NaHCO3 and NaCI absorption and is regulated rapidly by hemodynamic and neurohumoral factors to match the demands of the organism. Recent work from several laboratories have shown that NHE3 can be acutely regulated via a number O mechanisms. We have shown that hormones such as parathyroid hormone (PTH) and dopamine (DA) acutely inhibits NHE3 transport activity without changing its protein abundance on the plasma membrane. This inhibition is associated with complex changes in NHE3 phosphorylation and clustering of NHE3 proteins. We hypothesize that changes in NHE3 phosphorylation leads to NHE3 clustering which results in inhibition of activity. In this proposal. we will focus on: 1. Mapping out the amino acid residues n NHE3 that is regulated. 2. Determine whether changes in NHE3 phosphorylation per se alters its activity. 3. Determine whether NHE3 clustering is a consequence of changes in NHE3 phosphorylation and whether NHE3 clustering alters its activity. 4. Determine the role of two NHE3 binding proteins in mediating NHE3 clustering and changes in phosphorylation. We will apply protocols in transport physiology, biochemistry, recombinant DNA technology, immunohistology, and biophysical fluorescence spectroscopy to study whole animals, culture cells, and purified proteins. These studies will uncover highly fundamental mechanisms of regulation of an important epithelial Na transporter and further our understanding 01 disturbance in acid-base balance, Na retention and hypertension.