Regulation of Renal H+ATPase by Glycolysis

描述（由申请方提供）：肾脏酸分泌对于正常的酸基稳态、骨骼生长和肌肉代谢至关重要。异常尿液酸化是导致急、慢性肾脏疾病、肾结石和电解质紊乱发病的重要因素。肺泡H+ ATP酶（V-ATP酶）是由ATP驱动的产电质子泵，负责1/3或更多的近端小管质子分泌，并负责大部分集合管酸分泌。酸分泌与细胞代谢相关联，但对H+ ATP酶的代谢控制机制知之甚少。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酶与醛缩酶共定位于近端小管的顶膜，并且H+ ATP酶与Adolase和其他糖酵解酶的相互作用的破坏对H+ ATP酶具有重要的生理作用。在本申请中包括的初步数据中，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。 本提案的长期目标是研究H+ ATP酶与醛缩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。本研究的具体目的是：1）利用H + ATP酶亚基-融合蛋白构建体的结合试验，鉴定和表征H+ ATP酶E、B1和a4亚基上的醛缩酶结合位点; 2）通过研究醛缩酶与V-ATP酶结合的决定因素和酶促作用，研究醛缩酶与V-ATP酶的体外相互作用的功能意义; 3）通过表达正常和突变形式的醛缩酶，研究V-ATP酶-醛缩酶相互作用在完整肾上皮细胞中的功能意义和调节，以研究H+ ATP酶功能的生理效应。这些研究应导致重要的新的见解控制的肾脏酸分泌，对耦合的离子转运代谢，并对机制，有助于异常的葡萄糖代谢和糖尿病并发症。

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.