MOLECULAR BASIS OF RENAL URATE TRANSPORT

肾脏在维持尿酸盐中起着至关重要的作用。 动态平衡，为消除尿酸盐提供了主要途径 身体。虽然尿酸盐转运的两种机制，电子中和 阴离子交换和生电单端口已经被很好地描述， 这两个运输者的身份和特征都没有在 分子水平。这项提议的长期目标是理解 尿酸盐跨肾转运的分子机制 肾小管细胞膜。我们最近克隆了一个全长的cdna。 (命名为UAT)，从UAT cDNA制备重组蛋白，以及 重组蛋白在平面脂质中的功能重组 双分子膜作为尿酸盐转运体/通道。本文件中详细介绍的研究 该提案将检验这样一个假设，即我们拥有的独特的cdna 克隆的作为完整细胞膜的转运体/通道， 在肾脏内起到电生尿酸转运体的作用 这对尿酸盐的排泄有重要作用。四个具体目标 已经被开发出来检验这一假说。第一个目标是获得更多 广泛表征由编码的尿酸盐转运体/通道 重组蛋白在平面上融合后的UAT基因 脂质双分子层：通道的选择性，及其影响 PH、巯基、钙和磷酸化对通道活性的影响 将会被检查。第二个目标是评估和描述 UAT编码的尿酸盐转运体/通道的生物活性 转录后的基因在完整细胞中的表达 包括非洲爪哇的卵母细胞和永生化的人类肾脏细胞： 将进行两项微电极电压钳和膜片钳研究 受雇的。第三个目的是修改UAT的编码序列 CDNAs及其与功能相关的结构改变 编码蛋白质的活性。基因突变对细胞功能的影响 UAT中的特定氨基酸将通过电生理进行评估 平面脂质双层和/或卵母细胞的技术。最终目标是 以确定细胞和亚细胞内表达的部位 沿肾单位长度的UAT基因。这些研究将利用 原代培养和永生化细胞从定义的片段 人类肾脏的肾单位。

The kidneys play an essential role in the maintenance of urate homeostasis, providing the major route for elimination of urate from the body. Although two mechanisms of urate transport, electroneutral anion exchange and electrogenic uniport have been well described, neither transporter has been identified and characterized at the molecular level. The long term goal of this proposal is to understand the molecular mechanisms by which urate is transported across renal tubular cell membranes. We have recently cloned a full-length cDNA (designated UAT), prepared recombinant protein from the UAT cDNA, and functionally reconstituted the recombinant protein in planar lipid bilayers as a urate transporter/channel. The studies detailed in this proposal will test the hypothesis that the unique cDNA that we have cloned serves as a transporter/channel in intact cell membranes, functioning within the kidney as the electrogenic urate transporter that contributes importantly to urate excretion. Four specific aims have been developed to test this hypothesis. The first aim is to more extensively characterize the urate transporter/channel encoded by the UAT cDNA subsequent to fusion of the recombinant protein in planar lipid bilayers: the selectivity of the channel, and the influence of pH, sulfhydryl groups, calcium, and phosphorylation on channel activity will be examined. The second aim is to evaluate and characterize the biologic activity of the urate transporter/channel encoded by the UAT cDNA subsequent to its translation and expression in intact cells including Xenopus laevis oocytes and immortalized human renal cells: two microelectrode voltage clamp and patch clamp studies will be employed. The third aim is to modify the coding sequence of the UAT cDNA and correlate alterations in structure with the functional activity of the encoded protein. Functional effects of mutations in specific amino acids in UAT will be evaluated with electrophysiologic techniques in planar lipid bilayers and/or oocytes. The final aim is to characterize the cellular and subcellular sites of expression of the UAT cDNA along the length of the nephron. These studies will utilize primary cultures and immortalized cells from defined segments of the nephron of the human kidney.