Regulation of proton pump trafficking in kidney

肾脏质子泵运输的调节

• 批准号: 9049485
• 负责人: Dennis Brown
• 金额: $ 39.65万
• 依托单位: MASSACHUSETTS GENERAL HOSPITAL
• 依托单位国家: 美国
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-08-01 至 2018-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9049485
• 关键词: Acidosis; Acids; Adenylate Cyclase; Antibodies; Award; Biological Assay; Cell Culture System; Cells; Complex; Cyclic AMP; Cytoskeletal Proteins; Defect; Development; Distal; Epithelial Cells; Event; Fluorescence Resonance Energy Transfer; Genetic Transcription; Hormonal; In Vitro; Intercalated Cell; Kidney; Knockout Mice; Mass Spectrum Analysis; Measurement; Mediating; Membrane; Mus; Nephrolithiasis; Paper; Pathway interactions; Phosphorylation; Phosphorylation Site; Physiological; Process; Progress Reports; Proteins; Proteome; Proteomics; Proton Pump; Protons; Publishing; RNA Splicing; Regulation; Renal tubule structure; Role; Signal Pathway; Signal Transduction; Stimulus; Techniques; Time; Transgenic Mice; Tubular formation; Variant; Work; base; collecting tubule structure; in vivo; intravital microscopy; protein transport; response; tool; trafficking; vacuolar H+-ATPase

Defects in V-ATPase-dependent acidification in the kidney collecting duct results in distal tubular acidosis and nephrolithiasis. This MERIT award focuses on the role of intercalated cells (IC) in modulating proton secretion via the V-ATPase. Since the start of this award in 2008, we have published 20 original papers and 4 review articles on V- ATPase function and protein trafficking. Our work, summarized in the progress report, includes identifying signal transduction and trafficking pathways regulating proton- secretion by IC; identifying new V-ATPase interacting proteins; revealing many new splice variants of V-ATPase subunits that may have cell specific roles; performing an exhaustive proteomic analysis of 10; characterizing accessory proteins involved in trafficking pathways in different kidney tubules; generating new knockout and transgenic mice that open new avenues for understanding IC development and V-ATPase function; identifying multiple phosphorylation sites on V-ATPase subunits by mass spectrometry. Importantly, we have developed techniques to maintain differentiated 10 in vitro for several days. This now allows us unprecedented access to their proteome, RNA expression, and their acidification function in direct response to physiological/hormonal stimuli. In the extension period, we will: 1) Exterid our ongoing studies to identify the sensing and signal transduction cascade that leads to modulation of acid/base transport by 10 in the kidneys of normal and V-ATPase subunit-depJeted mice, and in our isolated EGFP-IC in vitro. We will use V-ATPase-Cre mice to selectively delete critical proteins including the soluble adenylyl cyclase (sAO), cytoskeletal proteins and accessory trafficking proteins from ICs. In vitro real time and in vivo intravital microscopy as well as functional measurements of proton secretion will be applied to follow 10 responses to stimuli. 2) We will dissect protein interactions and phosphorylation events that regulate V-ATPase trafficking and function a) by continuing to characterize cytoskeletal protein interactions (including FRET and Biacore assays) and b) by identifying phosphorylation events necessary for V-ATPase activation using expression of phosphomutant subunits in our cell culture systems. We will continue producing anti-phospho-V-ATPase antibodies. 3) We will characterize the expression, distribution and function of newly- identified V-ATPase a4 subunit splice variants that could explain differential membrane targeting of V-ATPase in 10 and other cells. In summary, our proposed studies are focused on the application-of new tools that will ultimately allow us to therapeutically modify renal acidification process by understanding acid/base sensing and V-ATPase trafficking mechanisms.

V-ATPase依赖性酸化的缺陷在肾脏收集管道中导致远端 肾小管酸中毒和肾结石病。该优点奖的重点是 通过V-ATPase调节质子分泌的插入细胞（IC）。自开始 该奖项在2008年，我们发表了20篇有关V-的原始论文和4篇评论文章。 ATPase功能和蛋白质运输。我们的工作，总结在进度报告中 包括识别信号转导和运输途径，以调节质子 IC分泌；识别新的V-ATPase相互作用蛋白；揭示了许多新的 可能具有细胞特定角色的V-ATPase亚基的剪接变体；执行 10的详尽蛋白质组学分析；表征涉及的配件蛋白 不同肾小管的贩运途径；产生新的淘汰和转基因 为理解IC开发和V-ATPase功能的新途径开放的小鼠； 通过质谱识别V-ATPase亚基上的多个磷酸化位点。 重要的是，我们开发了维护分化的10个体外技术的技术 几天。现在，这使我们可以空前访问其蛋白质组RNA 表达及其酸化功能在对生理/激素的直接反应中 刺激。在扩展期间，我们将：1）审查我们正在进行的研究以确定 传感和信号转导级联，导致酸/碱转运的调节 在正常和V-ATPase亚基消耗的小鼠的肾脏中的10个，在我们的孤立 EGFP-IC体外。我们将使用V-ATPase-Cre小鼠有选择地删除关键蛋白 包括可溶性腺苷酸环化酶（SAO），细胞骨架蛋白和附件 来自IC的运输蛋白质。体外实时和体内插入式显微镜以及 质子分泌的功能测量将应用于遵循10个对 刺激。 2）我们将剖析调节的蛋白质相互作用和磷酸化事件 V-ATPase运输和功能a）继续表征细胞骨架蛋白 通过鉴定磷酸化，相互作用（包括FRET和BIACORE分析）和B） 使用磷化剂亚基的表达进行V-ATPase激活所必需的事件 在我们的细胞培养系统中。我们将继续产生抗磷酸-V-ATPase 抗体。 3）我们将表征新的表达，分布和功能 确定的V-ATPase A4亚基剪接变体可以解释差异膜 在10和其他细胞中靶向V-ATPase。总而言之，我们提出的研究是 专注于新工具的应用，最终使我们能够治疗 通过了解酸/碱感应和V-ATPase来修改肾脏酸化过程 贩运机制。