Regulation of Renal H+ATPase by Glycolysis

描述（由申请人提供）：肾酸分泌对于正常的基于酸的体内平衡、骨骼生长和肌肉代谢至关重要。异常的尿酸化是导致急慢性肾脏疾病、肾结石和电解质紊乱的重要因素。液泡H+ atp酶（v - atp酶）是电致atp驱动的质子泵，负责近端小管1/3或更多的质子分泌，并负责大部分收集管酸分泌。酸分泌与细胞代谢相关，但H+ atp酶的代谢控制机制尚不清楚。我们最近发现液泡H+ atp酶的E亚基直接与糖酵解酶醛缩酶结合。我们证明了v - atp酶与醛缩酶在近端小管的顶膜上共定位，并且H+ atp酶与醛缩酶和其他糖酵解酶的相互作用的破坏对H+ atp酶有重要的生理影响。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 - atp酶质子分泌的有效激活剂，并且H+ atp酶的几个亚基与醛缩酶结合。

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.