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: 6201953
• 负责人: ARTHUR M BROWN
• 金额: $ 12.08万
• 依托单位: CASE WESTERN RESERVE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-09-01 至 2000-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6201953
• 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的突变产生了一种新的钾离子通道。 表现为低钾血症的产前变异型Bartter综合征 碱中毒、多尿和低血压。我们已经证明ROMK突变 减少K+通量，并提出这是该疾病的发病机制。一 一个必然的结果是，ROMK阻滞剂应该作为利尿剂和我们的长期 我们的目标是开发这种阻滞剂。我们的具体目标是：1）表征 产生Bartter综合征的ROMK突变，以确定重要的 蛋白质中的功能结构域并设计突变特异性疗法; 2)使用噬菌体展示产生调节ROMK的肽配体 通道功能，并将作为ROMK标签;以及3）确定 使用抗体或高亲和力抗体检测肾脏中ROMK糖基化状态 我们将开发的亲和配体。目标3源于我们的发现， 未糖基化ROMK中的K+电流显著降低。该项目将 不仅为巴特氏病的ROMK变体提供治疗， 提供了一类新的髓袢利尿剂。 设计实验来测试ROMK突变对K+的影响。 电流和组装，运输，磷酸化和蛋白水解 ROMK频道我们将把功能变化映射到拓扑模型 我们已经开发出的ROMK的糖基化位点插入 诱变为了发现ROMK特异性肽配体，我们将筛选 噬菌体展示文库。到 提供另一个理由改变ROMK电流，我们将研究 使用生物化学和免疫组织化学方法检测肾细胞中ROMK的糖基化 免疫细胞化学方法。 研究方法包括：重组DNA工程Bartter's 突变体，重组蛋白在草地贪夜蛾（Sf 9）中的表达 细胞;钾离子电流的膜片钳测量;生物化学方法 利用生物淘选技术筛选噬菌体展示文库; 测序分离的克隆;免疫细胞化学定位 肾和Sf 9细胞中的ROMK蛋白。