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 在调节流体和电解质传输中发挥重要作用 肾。 肾脏钾频道romki及其同工型ROMK2的cDNA 和3被隔离。 钾通道的功能障碍是 Bartter综合征的遗传原因。 我们的长期目标是 了解肾钾通道的分子组成， 通过饮食钾的摄入量调节钾通道 激素，调节通道的结构元素 磷脂酰肌醇4.5-双磷酸（PIP2）和分子基础 Bartter综合征中的通道功能障碍。 肾脏中的钾通道 由或不带有或不带有或/或不带有或/或不带有或没有 辅助蛋白囊性纤维化跨膜调节剂（CFTR）。 这 将研究肾脏钾通道的分子组成 大鼠肾脏的免疫组织化学共定位和免疫沉淀 组织。 ROMK多聚体和Romk-CFTR互动的化学计量学 将通过FPLC凝胶滤清色谱和蔗糖密度来检查 离心。 大鼠将保持在控制或高钾饮食中 在2周内检查饮食钾摄入量对ROMK的影响 表达。 确定醛固酮在ROMK调节中的作用 通过高钾的摄入表达，大鼠将被肾上腺切除术，并且 维持在控制或高钾饮食中，有或没有醛固酮 替代品。 Romk的结构要素参与了该法规 PIP2将通过爪蟾卵母细胞中的表达研究来检查。 这 Romk的细胞质结构域中充满电的残留物将是 由谷氨酰胺代替。 突变通道将在卵母细胞中表达 通过巨型研究，切除了内而外的贴片，以通过PIP2进行调节。 这 天然突变通道的细胞内处理和成熟 Bartter的综合征将通过培养的脉冲练习实验研究 细胞和胰腺微粒体的体外翻译。 结构 将检查由Bartter突变赋予的功能约束 通过筛选由饱和诱变构成的romk的库 钾摄取缺陷酵母菌菌株。