CHARACTERIZATION OF THE NA+-DRIVEN ANION EXCHANGER,NDAE1

调节细胞内和细胞外的pH值（酸碱运输），以及其他离子浓度，如Cl-和Na+，是维持跨膜离子梯度的关键。此外，正常的细胞功能是这些离子向内和向外运动之间的平衡，通常随细胞内ph值的变化而变化。这在中枢神经系统、消化道、呼吸道和泌尿系统中尤其如此。在过去几年中，已经克隆了几种离子转运蛋白cdna。近年来，我们利用表达克隆技术对肾电致Na/HCO3共转运体（NBC）进行了克隆和表征。我们现在克隆并表达了另一种新的酸碱转运体，即来自果蝇的Na+驱动的Cl-HCO3交换体。这种转运蛋白在生理学上已在神经元、肌肉、成纤维细胞和某些上皮细胞中发现。在肾脏中，这种Na+驱动的Cl-HCO3交换在成纤维细胞、系膜细胞和近端小管中起作用。目前为止，Na+驱动的Cl-HCO3交换器的作用似乎是调节细胞内ph。我们的表达数据表明，Na+驱动的Cl-HCO3交换器实际上是Na+驱动的阴离子交换器（NDAE1）。我们已经分离了几个哺乳动物克隆，人类ndae1基因，并开发了一个ndae1抗体。大鼠肾脏初步免疫定位显示肾小球、远端和集束管以及内髓小管明显染色。我们假设NDAE1在肾脏细胞内pH、Cl-和Na+的调节中起关键作用。为了验证这一假设，我们提出了三个目标：首先，为了确定NDAE1存在于哪些组织中，我们将克隆哺乳动物肾脏NDAE1同源物，并产生针对果蝇和哺乳动物蛋白质的抗体。结合Northern分析、Western分析和免疫化学，我们将确定哪些细胞和组织表达NDAE1。比较这两种生物体将有助于我们确定果蝇是否可能是肾脏离子运输的有用模型。其次，我们将通过在非洲爪蟾卵母细胞中的表达来研究NDAE1的分子生理。使用微电极技术的组合，我们将测量细胞内离子（H+， Cl-和Na+），电压钳，和离子电泳控制细胞离子。第三，我们将利用NDAE1片段的功能互补来确定NDAE1蛋白的重要结构域。由于我们有一个与阳离子相互作用的候选区域，我们将通过交换NBC的这个同源结构域和阴离子交换剂来扩大我们的结构域调查。这种方法的组合，应用于新克隆的Na+驱动阴离子交换器，将使我们能够确定NDAE1如何以及在何处调节肾脏细胞内pH， Cl-和Na+。

Regulation of intracellular and extracellular pH (acid-base transport), as well as other ionic concentrations, such as Cl- and Na+, are key to maintaining ion gradients across membranes. Moreover, normal cell function is a balance between inward and outward movement of these ions, often varying in response to intracellular pH. This is especially true in the central nervous system, digestive tract, respiratory tract, and urinary system. Several ion-transporter protein cDNAs have been cloned in the last few years. Recently, we used expression cloning to clone and characterize the renal electrogenic Na/HCO3 cotransporter (NBC). We have now cloned and expressed another novel acid-base transporter, the Na+ driven Cl-HCO3 exchanger from Drosophila. This transporter has been physiologically identified in neurons, muscle, fibroblasts, and certain epithelia. In the kidney, this Na+ driven Cl-HCO3 exchanger functions in fibroblasts, mesangial cells, and the proximal tubule. So far the role of the Na+ driven Cl-HCO3 exchanger appears to be regulation of intracellular pH. Our expression data indicate that the Na+ driven Cl-HCO3 exchanger is actually a Na+ driven anion exchanger (NDAE1). We have isolated several mammalian clones, the human NDAE1-gene, and developed a NDAE1-antibody. Preliminary immunolocalization in rat kidney shows pronounced staining in the glomerulus, distal and collecting ducts, and tubules of the inner medulla. We hypothesize the NDAE1 plays a key role in the regulation of intracellular pH, Cl-, and Na+ in the kidney. To test this hypothesis, we propose three aims: First, to determine in which tissues NDAE1 exists, we will clone the mammalian renal NDAE1 homologue and generate antibodies against both the Drosophila and mammalian proteins. Using a combination of Northern analysis, Western analysis, and immunochemistry, we will determine which cells and tissues express NDAE1. Comparing both organisms will help us determine if Drosophila might be a useful model for renal ion transport. Second, we will study the molecular physiology of NDAE1 by expression in Xenopus oocytes. Using a combination of microelectrode techniques, we will measure intracellular ions (H+, Cl-, and Na+), voltage clamp, and iontophoretically control cellular ions. Third, we will use functional complementation of NDAE1-fragments to determine structurally important domains of the NDAE1 protein. Since we have a candidate region for interaction with cations, we will augment our domain survey by exchanging this homologous domain of NBC and the anion exchangers. This combination of approaches, applied to the newly cloned Na+ driven anion exchanger, will allow us to determine how and where NDAE1 regulates intracellular pH, Cl-, and Na+ in the kidney.