CHARACTERIZATION OF THE NA+-DRIVEN ANION EXCHANGER,NDAE1

调节细胞内和细胞外的pH(酸碱转运)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，通常会随着细胞内pH的变化而变化。在中枢神经系统、消化道、呼吸道和泌尿系统尤其如此。在过去的几年里，已经克隆了几个离子转运蛋白的cDNA。最近，我们利用表达克隆的方法克隆和鉴定了肾脏生电Na/HCO3共转运蛋白(NBC)。我们现在已经克隆并表达了另一种新的酸碱转运蛋白--果蝇的Na+驱动的氯-HCO3交换器。这种转运蛋白已经在神经元、肌肉、成纤维细胞和某些上皮细胞中得到了生理学鉴定。在肾脏中，这种Na+驱动的氯-HCO3交换器在成纤维细胞、系膜细胞和近端小管中发挥作用。到目前为止，Na+驱动的氯-HCO3交换器的作用似乎是调节细胞内的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，人的NDAE1-基因，并开发了NDAE1-抗体。在大鼠肾脏的初步免疫定位显示，在肾小球、远端和集合管以及内髓的小管上有明显的染色。我们推测NDAE1在调节肾脏细胞内pH、Cl-和Na+方面起关键作用。为了验证这一假设，我们提出了三个目标：第一，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。比较这两种生物将有助于我们确定果蝇是否可能是肾脏离子转运的有用模型。其次，我们将通过在非洲爪哇卵母细胞中的表达来研究NDAE1的分子生理学。结合使用微电极技术，我们将测量细胞内离子(H+、Cl-和Na+)、电压钳和离子渗透控制细胞离子。第三，我们将使用NDAE1-片段的功能互补来确定NDAE1蛋白的结构重要结构域。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和阴离子交换器的这个同源结构域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+驱动的阴离子交换器，将使我们能够确定NDAE1如何以及在哪里调节肾脏细胞内的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.