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: 6105855
• 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: 1998
• 资助国家: 美国
• 起止时间: 1998-09-01 至 1999-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6105855
• 关键词: Bartter's syndrome; SDS polyacrylamide gel electrophoresis; Sf9 cell line; animal genetic material tag; autoradiography; confocal scanning microscopy; diuretics; drug design /synthesis /production; gene expression; gene mutation; gene targeting; glycosylation; immunoaffinity chromatography; immunocytochemistry; immunologic assay /test; kidney disorder chemotherapy; membrane structure; nucleic acid sequence; phosphorylation; polymerase chain reaction; potassium channel; protein structure function; renal medulla; voltage /patch clamp; western blottings

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.

肾外髓质K+通道ROMK突变导致 表现为低血钾的产前巴特综合征变异型 碱中毒，多尿和低血压。我们已经证明ROMK突变 减少K+流量，并提出这是该疾病的发病机制。一个 结论是ROMK阻滞剂应该起到利尿剂的作用，而且我们的长期 目标是开发这样的阻滞剂。我们的具体目标是：1)塑造 引起巴特综合征的ROMK突变对识别重要 蛋白质中的功能结构域，并设计突变特异性治疗； 2)利用噬菌体展示技术产生调节ROMK的多肽配体 通道功能，并将用作ROMK标签；以及3)确定 ROMK在肾脏中的糖基化状态 我们将开发的亲和配体。目标3源于我们的发现 未糖基化的ROMK的K+电流明显降低。该项目将 不仅为巴特的ROMK变种提供治疗，而且还将 提供一类新的环状利尿剂。 实验旨在测试ROMK突变对K+的影响 电流及其组装、运输、磷酸化和蛋白分解 ROMK频道。我们将把功能更改映射到一个拓扑模型 我们利用糖基化位点插入开发的ROMK 诱变。为了发现ROMK特异性的多肽配体，我们将筛选 用表达ROMK1的细胞进行生物扫描的噬菌体展示文库。至 提供了改变ROMK电流的另一个理由，我们将研究 肾细胞ROMK糖基化的生化和免疫组化研究 免疫细胞化学方法。 研究方法包括：重组DNA工程巴特氏病 突变型重组蛋白在果翅夜蛾中的表达 细胞.钾电流的膜片钳测量.生化方法 蛋白质分析；生物扫描筛选噬菌体展示文库； 对分离克隆进行测序；免疫细胞化学定位 ROMK蛋白在肾脏和Sf9细胞中的表达。