MOLECULAR MECHANISMS OF PROXIMAL TUBULE ION TRANSPORT

该项目的总体重点仍然是结构、功能 和生理作用的蛋白质介导的Na+-H+交换， 近曲小管细胞的质膜。编码四个Na+-H+的cDNA 已从哺乳动物细胞中克隆了交换器同种型（NHE 1 -4）。 所有四种同种型的转录物都在肾脏中表达。期间 过去的项目期间，我们产生了同种型特异性抗血清，以证明 NHE 1在基底外侧膜上表达，而NHE 3在基底外侧膜上表达。 表达于近曲小管细胞刷状缘膜。基于 在这个最初鉴定的NHE亚型表达在近端 小管细胞，我们将追求三个相互关联的目标，以表征 近端小管中NHE蛋白的结构和功能， 沿着肾单位。第一个总体目标是绘制细胞 和NHE同种型表达的亚细胞位点。为此我们 将继续产生同种型特异性的多克隆和单克隆 抗体的这些抗体将用于确定细胞和 近端小管中各亚型表达的膜位点， 沿着肾单位的免疫荧光和免疫电镜观察 显微镜下，并通过膜级分的蛋白质印迹分析。我们将 还评估了NHE亚型表达的时间和空间位点 新生儿肾脏发育中的肾单位。第二个总目标 是表征NHE亚型的重要功能， 特性.将在稳定转染的LAP 1细胞系中研究转运 表达每种同种型（NHE 1 -4）的Sf 9细胞，以及感染 编码每种同种型的重组杆状病毒。第三个总目标是 评估NHE亚型的结构与功能的关系。我们 将证实NHE 1和NHE 3各自是寡聚体的组分， 复合物，并将表征这些复合物的组成， 参与其生物合成和组装的步骤。的拓扑 NHE 3将通过抗肽抗体的定位和通过 刷状缘膜囊泡的载体蛋白水解。结构- NHE蛋白两亲性结构域内的功能关系 将通过使用嵌合构建体进行研究，以鉴定 确定发现同种型之间不同的功能特性，并通过 可能参与结合的特定氨基酸残基的突变 和/或阳离子和H+的运输。 关于NHE同种型的分子特性及其 沿着肾单位的表达位点将提供对 NHE同种型在整合的肾功能中的生理作用， HCO 3-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.