RENAL POTASSIUM TRANSPORT IN PHYSIOLOGY AND DISEASES

生理学和疾病中的肾脏钾转运

• 批准号: 6381209
• 负责人: Chou-Long Huang
• 金额: $ 23.81万
• 依托单位: UT SOUTHWESTERN MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-08-01 至 2002-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6381209
• 关键词: Bartter's syndrome; Xenopus oocyte; adrenalectomy; aldosterone; density gradient ultracentrifugation; dietary potassium; gel filtration chromatography; gene mutation; genetic library; glutamine; immunocytochemistry; immunoprecipitation; laboratory rabbit; laboratory rat; microsomes; molecular pathology; nutrition related tag; phosphatidylinositols; potassium channel; protein isoforms; protein localization; protein structure function; renal tubular transport; tissue /cell culture; yeasts

DESCRIPTION (Adapted from the Applicant's Abstract): Potassium channels play essential roles in the regulation of fluid and electrolyte transport in kidney. cDNAs for the renal potassium channel ROMKI and its isoforms ROMK2 and 3 have been isolated. Dysfunction of the potassium channels is one of the genetic causes for Bartter's syndrome. Our long-term objectives are to understand the molecular composition of the renal potassium channels, the regulation of the potassium channels by dietary potassium intake and by hormones, the structural elements of the channel for regulation by phosphatidylinositol 4.5-bisphosphate (PIP2), and the molecular basis for channel dysfunction in Bartter's syndrome. Potassium channels in kidney may consist of hetero- and/or homo-multimers of ROMK isoforms with or without accessory proteins cystic fibrosis transmembrane regulator (CFTR). The molecular composition of the renal potassium channels will be studied using immunohistochemical co-localization and co-immunoprecipitation of rat kidney tissue. The stoichiometry of ROMK multimers and of ROMK-CFTR interaction will be examined by FPLC gel-filtration chromatography and sucrose-density centrifugation. Rats will be maintained in control or high potassium diets for 2 weeks to examine the effect of dietary potassium intake on ROMK expression. To determine the role of aldosterone in the regulation of ROMK expression by high potassium intake, rats will be adrenalectomized and maintained in control or high potassium diet with or without aldosterone replacement. The structural elements of ROMK involved in the regulation by PIP2 will be examined by expression studies in Xenopus oocytes. The positively charged residues in the cytoplasmic domain of ROMK will be replaced by glutamine. Mutant channels will be expressed in oocytes and studied by giant excised inside-out patches for regulation by PIP2. The intracellular processing and maturation of the natural mutant channels of Bartter's syndrome will be studied by pulse-chase experiments in cultured cells and by in vitro translation in pancreatic microsomes. The structural and functional constraints conferred by Bartter's mutation will be examined by screening libraries of ROMK constructed by saturation mutagenesis in a potassium uptake-defective yeast strains.

描述（改编自申请人的摘要）：钾通道 在体液和电解质运输的调节中发挥重要作用 肾。 肾钾通道 ROMKI 及其亚型 ROMK2 的 cDNA 3人已被隔离。 钾通道功能障碍是其中之一 巴特综合症的遗传原因。 我们的长期目标是 了解肾钾通道的分子组成 通过膳食钾摄入量和钾通道的调节 激素，调节通道的结构元件 磷脂酰肌醇 4.5-二磷酸 (PIP2) 及其分子基础 巴特综合征中的通道功能障碍。 肾脏中的钾通道可能 由ROMK亚型的异源和/或同源多聚体组成，有或没有 辅助蛋白囊性纤维化跨膜调节蛋白（CFTR）。 这 将使用以下方法研究肾钾通道的分子组成 大鼠肾脏的免疫组化共定位和免疫共沉淀 组织。 ROMK 多聚体和 ROMK-CFTR 相互作用的化学计量 将通过 FPLC 凝胶过滤色谱法和蔗糖密度进行检查 离心。 大鼠将维持对照饮食或高钾饮食 为期 2 周，检查膳食钾摄入量对 ROMK 的影响 表达。 确定醛固酮在 ROMK 调节中的作用 通过高钾摄入表达，大鼠将被切除肾上腺并 维持控制饮食或高钾饮食，含或不含醛固酮 替代品。 ROMK参与调节的结构元件 PIP2 将通过非洲爪蟾卵母细胞中的表达研究进行检查。 这 ROMK 胞质结构域中带正电荷的残基将是 被谷氨酰胺取代。 突变通道将在卵母细胞中表达 通过巨大的切除的由内而外的补丁来研究 PIP2 的调节。 这 自然突变通道的细胞内加工和成熟 巴特综合症将通过脉冲追踪实验在培养物中进行研究 细胞并通过胰腺微粒体的体外翻译。 结构性 将检查 Bartter 突变所赋予的功能限制 通过筛选通过饱和诱变构建的ROMK文库 钾吸收缺陷的酵母菌株。