MOLECULAR MECHANISMS OF PROXIMAL TUBULE ION TRANSPLANT

这个项目的总体重点仍然是结构、功能 以及介导Na+-H+交换的蛋白质(S)的生理作用 近端小管细胞的质膜。编码四种Na+-H+的cDNA 交换异构体(NHE1-4)已从哺乳动物细胞中克隆出来。 所有四种亚型的转录本都在肾脏中表达。在.期间 在过去的项目期间，我们生成了异构体特异的抗血清来证明 NHE1在基底膜上表达，而NHE3在基底膜上表达 表达于近端小管细胞的刷状缘膜上。基座 NHE异构体在近端表达的初步鉴定 小管细胞，我们将追求三个相互关联的目标来表征 近曲小管和近曲小管NHE蛋白的结构和功能 沿肾单位的其他地方。第一个总体目标是绘制细胞 以及NHE亚型的亚细胞表达部位。为此，我们 将继续产生特定于异构体的多克隆和单克隆 抗体。这些抗体将被用来确定细胞和 各亚型在近曲小管和近曲小管中的表达部位 免疫荧光和免疫电子技术在肾单位内的分布 显微镜观察，并通过Western印迹分析膜组分。我们会 同时评估NHE异构体表达的时间和空间位置 在新生儿肾脏的发育中的肾单位。第二个总目标 是根据重要的官能团来表征NHE亚型 属性。LAP1稳定转染系的转运将被研究 表达每种亚型(NHE1-4)的细胞，以及感染了NHE1-4的Sf9细胞 编码每个亚型的重组杆状病毒。第三个总目标是 评估NHE异构体的结构与功能的关系。我们 将确认NHE1和NHE3分别是齐聚物的组分 并将表征这些络合物的组成和 它们的生物合成和组装所涉及的步骤。的拓扑图 NHE3将通过抗肽抗体的定位和 刷状缘膜囊泡的矢量蛋白分解。结构- NHE蛋白两亲性结构域内的功能关系 将通过使用嵌合结构来研究，以识别 确定发现不同异构体之间的功能特性不同，并通过 可能参与结合的特定氨基酸残基突变 和/或阳离子和H+的运输。 关于NHE亚型及其亚型的分子性质的信息 沿肾单位的表达部位将提供对 NHE亚型在综合肾功能中的生理学作用 HCO3-重吸收和酸分泌。

The general focus of this project continues to be the structure, function and physiologic roles of the protein(s) mediating Na+-H+ exchange across the plasma membranes of proximal tubule cells. cDNAs encoding four Na+-H+ exchanger isoforms (NHE1-4) have been cloned from mammalian cells. Transcripts for all four isoforms are expressed in the kidney. During the past project period we generated isoform-specific antisera to demonstrate that NHE1 is expressed on the basolateral membrane whereas NHE3 is expressed on the brush border membrane of proximal tubule cells. Based on this initial identification of NHE isoforms expressed in proximal tubule cells, we will pursue three interrelated aims to characterize the structure and function of NHE proteins in the proximal tubule and elsewhere along the nephron. The first general aim is to map the cellular and subcellular sites of expression of NHE isoforms. For this purpose we will continue to generate isoform-specific polyclonal and monoclonal antibodies. These antibodies will be used to determine the cell and membrane sites of expression of each isoform in the proximal tubule and along the nephron by use of immunofluorescence and immunoelectron microscopy, and by Western blot analysis of membrane fractions. We will also assess the temporal and spatial sites of expression of NHE isoforms in the developing nephron of the neonatal kidney. The second general aim is to characterize NHE isoforms with respect to important functional properties. Transport will be studied in stably transfected lines of LAP1 cells that express each isoform (NHE1-4), and in Sf9 cells infected with recombinant baculovirus encoding each isoform. The third general aim is to assess the relationship of structure to function of NHE isoforms. We will confirm that NHE1 and NHE3 are each components of oligomeric complexes, and will characterize the composition of these complexes and the steps involved in their biosynthesis and assembly. The topology of NHE3 will be assessed by localization of anti-peptide antibodies and by vectorial proteolysis of brush border membrane vesicles. Structure- function relationships within the amphipathic domains of NHE proteins will be studied by use of chimeric constructs to identify subdomains that determine functional properties found to differ between isoforms, and by mutation of specific amino acid residues likely to participate in binding and/or transport of cations and H+. Information about both the molecular properties of NHE isoforms and their sites of expression along the nephron will provide insight into the physiologic roles of NHE isoforms in such integrated kidney functions as HCO3 - reabsorption and acid secretion.