MOLECULAR BASIS OF RENAL URATE TRANSPORT

肾脏尿酸盐转运的分子基础

• 批准号: 2906040
• 负责人: RUTH G ABRAMSON
• 金额: $ 25.59万
• 依托单位: ICAHN SCHOOL OF MEDICINE AT MOUNT SINAI
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-08-01 至 2001-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2906040
• 关键词: Xenopus oocyte; complementary DNA; gene expression; human tissue; lipid bilayer membrane; membrane channels; membrane transport proteins; molecular cloning; protein sequence; protein structure function; recombinant proteins; renal tubular transport; tissue /cell culture; urate

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.

肾脏在维持尿酸方面发挥着重要作用 体内平衡，提供消除尿酸盐的主要途径 身体。尽管尿酸盐转运有两种机制，电中性 阴离子交换和生电单端口已得到很好的描述， 两种转运蛋白均尚未被识别和表征 分子水平。该提案的长期目标是了解 尿酸跨肾转运的分子机制 管状细胞膜。我们最近克隆了全长 cDNA (命名为UAT)，从UAT cDNA制备重组蛋白，以及 在平面脂质中功能性重建重组蛋白 双层作为尿酸盐转运蛋白/通道。 本文详细介绍了这些研究 该提案将检验我们拥有的独特 cDNA 的假设 克隆作为完整细胞膜中的转运蛋白/通道， 在肾脏内作为生电尿酸盐转运蛋白发挥作用 这对尿酸盐的排泄有重要贡献。四个具体目标 已被开发来检验这一假设。第一个目标是更多 广泛表征由编码的尿酸盐转运蛋白/通道 平面重组蛋白融合后的 UAT cDNA 脂质双层：通道的选择性以及 pH、巯基、钙和磷酸化对通道活性的影响 将接受检查。 第二个目标是评估和表征 UAT 编码的尿酸盐转运蛋白/通道的生物活性 在完整细胞中翻译和表达后的 cDNA 包括非洲爪蟾卵母细胞和永生化人肾细胞： 两项微电极电压钳和膜片钳研究将 受雇。第三个目的是修改UAT的编码序列 cDNA 并将结构变化与功能相关联 所编码蛋白质的活性。 突变的功能影响 UAT 中的特定氨基酸将通过电生理学进行评估 平面脂质双层和/或卵母细胞技术。最终的目标是 表征细胞和亚细胞表达位点 沿着肾单位长度的 UAT cDNA。这些研究将利用 原代培养物和来自指定片段的永生化细胞 人类肾脏的肾单位。