Regulation of proton pump trafficking in kidney

肾脏质子泵运输的调节

基本信息

批准号：
8250029
负责人：
Dennis Brown
金额：
$ 36.66万
依托单位：
MASSACHUSETTS GENERAL HOSPITAL
依托单位国家：
美国
项目类别：
财政年份：
1991
资助国家：
美国
起止时间：
1991-08-01 至 2013-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8250029
关键词：
ATP phosphohydrolase Acid-Base Imbalance Acidosis Acids Actins Address Adenylate Cyclase Alkalosis Animal Model Bicarbonate Ion Bicarbonate Ions Bicarbonates Binding Biological Assay Blood Body Fluids Cell Culture Techniques Cell membrane Cell physiology Cells Chimera organism Complex Confocal Microscopy Cues Cyclic AMP Cyclic AMP-Dependent Protein Kinases Cytoskeletal Proteins Cytoskeleton Defect Detection Development Disease Dominant-Negative Mutation Electrodes Endocytosis Endosomes Energy Transfer Epididymis Epithelial Cells Equilibrium Event Excretory function Exocytosis Fluorescence Functional disorder Funding Gel Gelsolin Generations Goals Health Human Imaging technology In Situ In Vitro Intercalated Cell Kidney Lead Life Mass Spectrum Analysis Mediating Membrane Membrane Microdomains Microelectrodes Monitor Mutation Normal Cell Normal Range Organ Pathway interactions Phosphorylation Physiological Play Process Proline Protein Tyrosine Kinase Proteins Proteomics Proton Pump Protons Published Comment Recruitment Activity Regulation Role Second Messenger Systems Signal Pathway Signal Transduction Small Interfering RNA Sorting - Cell Movement System Time Translating Tubular formation Urine Vesicle Western Blotting Work Writing base body system cellular imaging collecting tubule structure computerized data processing drebrins extracellular genetic regulatory protein in vitro Assay induced pluripotent stem cell interdisciplinary approach myosin VI nadrin novel protein complex protein expression protein protein interaction response second messenger sensor trafficking vacuolar H+-ATPase

项目摘要

Intercalated cells (IC) respond to pH changes in the blood by increasing or decreasing acid secretion in the kidney collecting duct. Dysfunction of this process results in pathophysiological disorders of different organ systems as the pH of the blood drifts away from its normal value of 7.4. The vacuolar H[+]ATPase (V-ATPase) is central to the acid/base homeostatic function of IC, but how these cells detect environmental cues that allows them to modify proton secretion appropriately remains a mystery. The existence of an acid or bicarbonate sensor in the kidney has long been suggested, but the identity of this detection system and how such a system would transmit signals to modify the acid/base transporting machinery of ICs remain to be determined. Based on work carried out in the previous funding period, we propose here that the soluble adenylate cyclase (sAC) is the much sought after renal acid/base sensor. This protein generates the second messenger cAMP upon direct stimulation by bicarbonate ions. It is, therefore, ideally suited for a bicarbonate/CO{2} sensing role in IC. We hypothesize that cAMP generated by the sAC sensor in response to acid/base cues can modify the acid secretory capacity of intercalated cells. We propose that V-ATPase and sAC are partners in a localized signaling process that modulates targeting and trafficking of the V-ATPase in specific membrane microdomains to regulate intercalated cell function, and renal proton secretion. Our aims are: 1) To characterize the role of sAC in the regulation of V-ATPase mediated proton secretion by renal epithelial cells and 2) To determine whether V- ATPase and cytoskeletal proteins (actin, gelsolin, drebrin, nadrin and myosin VI) form a local micro-complex that regulates V-ATPase membrane accumulation and proton secretion in IC. The studies will use a multidisciplinary approach including unique animal models, isolated fluorescence-sorted intercalated cells, and cell cultures, as well as imaging technologies including static and real-time confocal microscopy to follow V- ATPase trafficking. Assays of vesicle acidification, ATPase activity and proton-selective self-referencing microelectrodes will monitor the functional expression of V-ATPase in endosomes and at the plasma membrane. Fluorescence (Forsman) resonance energy transfer (FRET) and protein-protein interaction assays will dissect whether V-ATPase subunits interact with sAC and/or cytoskeletal proteins during stimulation of proton secretion. In vitro assays, mutational analysis and phosphoproteomics will address the role of cAMP/PKA mediated V-ATPase phosphorylation in these interactions. We propose that the V-ATPase is a central partner in a localized, multi-protein complex that senses and responds to prevailing acid/base conditions by modulating the V-ATPase dependent acidification mechanism in intercalated cells.

插入的细胞（IC）通过增加或减少肾脏收集导管中的酸分泌来应对血液的pH变化。该过程的功能障碍会导致不同器官系统的病理生理疾病，因为血液的pH远离其正常值7.4。液泡H [+] ATPase（V-ATPase）对于IC的酸/碱基稳态功能是核心，但是这些细胞如何检测环境线索，使它们能够适当地修改质子分泌仍然是一个谜。长期以来，已经提出了肾脏中酸或碳酸氢盐传感器的存在，但是该检测系统的身份以及该系统将如何传输信号以修改ICS的酸/碱基运输机械。基于上一个融资期间进行的工作，我们在这里建议可溶性腺苷酸环化酶（SAC）是肾酸/碱基传感器的备受追捧的。该蛋白质在碳酸氢盐离子直接刺激后产生第二个信使训练营。因此，它非常适合IC中的碳酸氢盐/CO {2}传感作用。我们假设由SAC传感器生成的cAMP对酸/碱提示产生，可以改变插入细胞的酸分泌能力。我们建议V-ATPase和SAC是局部信号传导过程中的合作伙伴，该过程调节了特定膜微区域中V-ATPase的靶向和运输以调节介导的细胞功能和肾脏质子分泌。我们的目的是：1）表征SAC在肾上皮细胞对V-ATPase介导的质子分泌和2）中的作用，以确定V- ATPase和细胞骨架蛋白（肌动蛋白，凝胶蛋白，Dreberin，drebrin，Nadrin，Nadrin和Myosin vi）是否会累积v-atpase V-Atpase V-Atpase。这些研究将使用多学科方法，包括独特的动物模型，孤立的荧光分级插入的细胞以及细胞培养物，以及包括静态和实时共焦显微镜在内的成像技术，以遵循V- ATPase运输。囊泡酸化，ATPase活性和质子选择的自我引用微电极的测定将监测内体和质膜中V-ATPase的功能表达。荧光（Forsman）共振能量转移（FRET）和蛋白质 - 蛋白质相互作用测定将在刺激质子分泌过程中剖析V-ATPase亚基是否与SAC和/或细胞骨架蛋白相互作用。在体外测定中，突变分析和磷酸蛋白质组学将解决CAMP/PKA介导的V-ATPase磷酸化在这些相互作用中的作用。我们建议，V-ATPase是局部多蛋白质复合物中的核心伴侣，通过调节Intercalateclated细胞中的V-ATPase依赖性酸化机制，可以感知并响应现行的酸/碱条件。