TARGETING RENAL OUTER MEDULLARY K+ CHANNEL ROMK FOR NEW CLASS OF DIURETICS

针对肾外髓 K 通道 ROMK 的新型利尿剂

• 批准号: 6651771
• 负责人: ARTHUR M BROWN
• 金额: $ 13.53万
• 依托单位: CASE WESTERN RESERVE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-09-01 至 2003-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6651771
• 关键词: Bartter's syndrome; Sf9 cell line; animal genetic material tag; diuretics; drug design /synthesis /production; drug discovery /isolation; gene expression; gene mutation; gene targeting; glycosylation; ion channel blocker; kidney disorder chemotherapy; membrane structure; phosphorylation; polymerase chain reaction; potassium channel; protein structure function; renal medulla

Mutations in the renal outer medullary K+ channel ROMK produce an antenatal variant of Bartter's syndrome manifested by hypokalemic alkalosis, polyuria and hypotension. We have shown that ROMK mutations reduce K+ flux and propose this as the pathogenesis for the disease. A corollary is that ROMK blockers should act as diuretics and our long-term goal is to develop such blockers. Our specific aims are: 1) characterize the ROMK mutations that produce Bartter's syndrome to identify important functional domains in the protein and to design mutation-specific therapy; 2) use phage display to generate peptide ligands which regulate ROMK channel function and which will serve as ROMK tags; and 3) determine the status of ROMK glycosylation in kidney using either antibodies or high affinity ligands that we will develop. Aim 3 derives from our finding that K+ currents in un-glycosylated ROMK are markedly reduced. The project will not only provide therapy for the ROMK variant of Bartter's but will also provide a new class of loop diuretics. Experiments are designed to test the effects of ROMK mutations on K+ currents and assembly, trafficking, phosphorylation and proteolysis of ROMK channels. We will map the functional changes to a topological model of ROMK that we have developed using glycosylation site insertion mutagenesis. To discover ROMK-specific peptide ligands we will screen phage display libraries by biopanning with cells expressing ROMK1. To provide another rationale for altering ROMK currents we will examine the glycosylation of ROMK in kidney cells using biochemical and immunocytochemical methods. The research methods include: recombinant DNA to engineer Bartter's mutant, expression of recombinant proteins in Spodoptera frugiperda (Sf9) cells; patch-clamp measurements of K+ currents; biochemical methods for analysis of protein; screening phage display libraries by biopanning; sequencing isolated clones; and immunocytochemistry for localization of ROMK protein in kidney and Sf9 cells.

肾外髓 K+ 通道 ROMK 的突变会产生 巴特综合征的产前变异，表现为低钾血症 碱中毒、多尿和低血压。我们已经证明ROMK突变 减少 K+ 通量并提出将此作为该疾病的发病机制。一个 推论是 ROMK 阻滞剂应该起到利尿剂的作用，并且我们的长期 目标是开发这样的阻断剂。我们的具体目标是：1）表征 产生 Bartter 综合征的 ROMK 突变可识别重要的 蛋白质的功能域并设计突变特异性疗法； 2）利用噬菌体展示生成调节ROMK的肽配体 通道功能，将作为ROMK标签； 3) 确定 使用抗体或高水平检测肾脏中 ROMK 糖基化的状态 我们将开发的亲和配体。目标 3 源于我们的发现 未糖基化的 ROMK 中的 K+ 电流显着降低。该项目将 不仅为巴特氏症的 ROMK 变体提供治疗，而且还将 提供一类新的袢利尿剂。 实验旨在测试 ROMK 突变对 K+ 的影响 电流和组装、运输、磷酸化和蛋白水解 ROMK频道。我们将功能变化映射到拓扑模型 我们使用糖基化位点插入开发的 ROMK 诱变。为了发现 ROMK 特异性肽配体，我们将筛选 通过用表达 ROMK1 的细胞进行生物淘选来构建噬菌体展示文库。到 提供改变 ROMK 电流的另一个理由，我们将检查 使用生化和方法研究肾细胞中 ROMK 的糖基化 免疫细胞化学方法。 研究方法包括： 重组 DNA 来改造 Bartter's 突变体，草地贪夜蛾 (Sf9) 中重组蛋白的表达 细胞； K+ 电流的膜片钳测量；生化方法 蛋白质分析；通过生物淘选筛选噬菌体展示文库； 对分离的克隆进行测序；和免疫细胞化学定位 肾脏和 Sf9 细胞中的 ROMK 蛋白。