MOLECULAR BASIS OF RENAL URATE TRANSPORT

肾脏在维持尿酸盐中起着重要作用 体内平衡，提供消除尿酸盐的主要途径， 身体虽然尿酸盐转运的两种机制，电中性 阴离子交换和产电单端口已经被很好地描述， 这两种转运蛋白都没有在 分子水平。本提案的长期目标是了解 尿酸盐通过肾脏转运的分子机制 管状细胞膜我们最近克隆了一个全长cDNA （命名为UAT），从UAT cDNA制备重组蛋白，和 在平面脂质中功能性地重构重组蛋白 双层作为尿酸盐转运蛋白/通道。 这篇文章中详细介绍的研究 这项提案将检验一个假设，即我们拥有的独特cDNA cloned作为完整细胞膜中的转运蛋白/通道， 在肾脏内作为产电尿酸盐转运体发挥作用 对尿酸盐排泄有重要作用。四个具体目标 来验证这个假设第一个目标是更多 广泛表征尿酸转运蛋白/通道编码的 UAT cDNA在重组蛋白融合后的平面表达 脂质双层：通道的选择性，以及 pH、巯基、钙离子和磷酸化对通道活性的影响 将被审查。 第二个目的是评估和表征 由UAT编码的尿酸盐转运蛋白/通道的生物活性 cDNA随后在完整细胞中的翻译和表达 包括非洲爪蟾卵母细胞和永生化人肾细胞： 将进行两项微电极电压钳和膜片钳研究， 就业。第三个目的是修改UAT的编码序列 cDNA和相关的结构变化与功能 编码蛋白质的活性。 基因突变的功能效应 UAT中的特定氨基酸将用电生理学方法进行评价。 在平面脂质双层和/或卵母细胞中的技术。最终目标是 为了表征表达的细胞和亚细胞位点， UAT cDNA沿着肾单位的长度。这些研究将利用 原代培养物和永生化细胞从确定的节段， 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.