Regulation of Renal H+ATPase by Glycolysis

糖酵解对肾 H ATP 酶的调节

• 批准号: 6835617
• 负责人: STEPHEN L GLUCK
• 金额: $ 35.6万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-03-01 至 2007-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6835617
• 关键词: acidity /alkalinity; adenosinetriphosphatase; aldehyde lyase; binding sites; enzyme activity; enzyme biosynthesis; enzyme mechanism; enzyme structure; glycolysis; hydrogen transport; hydrogen transporting ATP synthase; kidney metabolism; laboratory mouse; protein protein interaction; renal tubular transport; site directed mutagenesis; tissue /cell culture

DESCRIPTION (provided by applicant): Renal acid secretion is essential for normal acid-based homeostasis, skeletal growth, and muscle metabolism. Abnormal urinary acidification is an important contributing factor in the morbidity of acute and chronic kidney disease, nephrolithiasis, and electrolyte disorders. Vacuolar H+ATPases (V-ATPases) are electrogenic ATP-driven proton pumps responsible for 1/3 or more of proximal tubule proton secretion, and for the majority of collecting duct acid secretion. Acid secretion is coupled to cellular metabolism, but the mechanisms for metabolic control of the H+ATPase are poorly understood. We recently discovered that the E subunit of the of the vacuolar H+ATPase binds directly to the glycolytic enzyme aldolase. We demonstrated that the V-ATPase colocalizes with aldolase at he apical membrane in the proximal tubule, and that disruption of the interaction of the H+ATPase with adolase and other glycolytic enzymes has important physiologic effects on the H+ATPase. In preliminary data included in this application, we demonstrate that glucose is a potent activator of V-ATPase proton secretion in cultured renal epithelial cells, and that several subunits of the H+ATPase bind to aldolase. The long-term objective of this proposal is to examine the structural basis and regulation of the interaction of the H+ATPase with aldolase, and its role in control of renal proton secretion. The Specific Aims are 1) to identify and characterize the aldolase binding site on the H+ATPase E, B1 and a4 subunits using binding assays with H+ATPase subunit-fusion protein constructs; 2) to examine the functional significance of the aldolase-V-ATPase interaction in vitro by studying the determinants and enzymatic effects of aldolase binding to the V-ATPase; and 3) to study the functional significance and regulation of the V-ATPase-aldolase interaction in intact renal epithelial cells by expressing normal and mutated forms of aldolase to study the physiologic effects on H+ATPase function. These studies should lead to important new insights on the control of renal acid secretion, on the coupling of ion transport to metabolism, and on mechanisms that contribute to abnormal glucose metabolism and complications of diabetes mellitus.

描述（由申请人提供）：肾脏酸分泌对于基于酸的正常稳态，骨骼生长和肌肉代谢至关重要。异常尿酸化是急性和慢性肾脏疾病，肾石石症和电解质疾病的发病率的重要因素。液泡H+ATPases（V-ATPases）是电源ATP驱动的质子泵，负责1/3或更多的近端小管质子分泌，并且大多数收集导管酸分泌。酸分泌与细胞代谢耦合，但是对H+ATPase的代谢控制的机制知之甚少。我们最近发现，液泡H+ATPase的E亚基直接与糖酵解酶醛糖酶结合。我们证明了V-ATPase与近端小管中HE顶膜的藻酶共定位，并且H+ATPase与adolase与adolase和其他糖酵解酶的相互作用的破坏具有重要的生理作用对H+ATPase。在本应用程序中包含的初步数据中，我们证明葡萄糖是培养的肾上皮细胞中V-ATPase质子分泌的有效活化剂，H+ATPase的几个亚基与醛糖酶结合。 该提案的长期目标是检查H+ATPase与醛酶相互作用的结构基础和调节，及其在控制肾脏质子分泌中的作用。具体目的是1）使用具有H+ATPase亚基融合蛋白构建体H+ATPase E，B1和A4亚基上醛糖酶结合位点的表征； 2）通过研究链氨酶与V-ATPase的决定因素和酶促作用，以检查体外醛糖酶-V-ATPase相互作用的功能意义； 3）通过表达正常和突变形式的醛糖酶形式来研究完整肾上皮细胞中V- ATPase-溶解酶相互作用的功能显着性和调节，以研究对H+ATPase功能的生理影响。这些研究应导致对肾酸分泌的控制，离子转运与新陈代谢的耦合以及有助于异常葡萄糖代谢和糖尿病并发症的机制的重要新见解。