Regulation of Renal H+ATPase by Glycolysis

描述(申请人提供)：肾酸分泌对正常的酸基平衡、骨骼生长和肌肉新陈代谢是必不可少的。尿酸异常是急、慢性肾脏疾病、肾结石和电解质紊乱的重要致病因素。液泡H+-ATPase(V-ATPase)是由ATP驱动的电生质子泵，负责近端小管1/3或更多的质子分泌，并负责收集管酸的大部分分泌。酸分泌与细胞代谢有关，但H+ATPase的代谢控制机制知之甚少。我们最近发现，液泡H+ATPase的E亚基直接与糖酵解酶醛缩酶结合。我们证明了V-ATPase与醛缩酶共定位于近端小管的顶膜，并且H+ATPase与多糖酶和其他糖酵解酶的相互作用的中断对H+ATPase有重要的生理作用。在本申请的初步数据中，我们证明葡萄糖是培养的肾上皮细胞V-ATPase质子分泌的有效激活剂，并且H+ATPase的几个亚基与醛缩酶结合。 这一建议的长期目标是研究H+-ATPase与醛缩酶相互作用的结构基础和调节，以及它在控制肾脏质子分泌中的作用。其具体目的是：1)利用与H+ATPase亚单位-融合蛋白构建物的结合分析，鉴定和表征H+ATPase E、B1和A4亚基上的醛缩酶结合部位；2)通过研究醛缩酶与V-ATPase结合的决定因素和酶作用，探讨醛缩酶-V-ATPase相互作用在体外的功能意义；以及3)通过表达正常和突变形式的醛缩酶，研究V-ATPase-醛缩酶相互作用在完整肾上皮细胞中的功能意义和调节，以研究其对H+ATPase功能的生理影响。这些研究将在控制肾酸分泌、离子转运与代谢的耦合以及导致糖代谢异常和糖尿病并发症的机制方面带来重要的新见解。

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.