Regulation of proton pump trafficking in kidney

肾脏质子泵运输的调节

• 批准号: 8072140
• 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
• 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/8072140
• 关键词: ATP phosphohydrolase; Acid-Base Imbalance; Acidosis; Acids; Actins; Address; Adenylate Cyclase; Alkalosis; Animal Model; Bicarbonate Ion; Bicarbonate Ions; Bicarbonates; Biological Assay; Blood; Body Fluids; Carbon Dioxide; Cell Culture Techniques; Cell membrane; Cell physiology; Cells; Complex; Confocal Microscopy; Cues; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Cytoskeletal Proteins; Detection; Development; Disease; Endosomes; Energy Transfer; Epithelial Cells; Equilibrium; Excretory function; Fluorescence; Functional disorder; Funding; Gelsolin; Health; Imaging technology; Intercalated Cell; Kidney; Mediating; Membrane Microdomains; Microelectrodes; Monitor; Normal Range; Phosphorylation; Physiological; Play; Process; Proteins; Proton Pump; Protons; Regulation; Role; Second Messenger Systems; Signal Transduction; Sorting - Cell Movement; System; Time; Urine; Vesicle; Work; base; body system; collecting tubule structure; computerized data processing; drebrins; in vitro Assay; interdisciplinary approach; myosin VI; nadrin; protein complex; protein protein interaction; response; second messenger; sensor; trafficking; vacuolar H+-ATPase

DESCRIPTION (provided by applicant): 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/CO2 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. Maintaining the acid/base (i.e., pH) level of body fluids, including the blood, within a narrow range is critical to normal health and to the function of all cells and organ systems. The kidney plays a central role in this process by sensing and eliminating excess acid or excess base via excretion into the urine. Currently, the sensing mechanism by which the kidney detects and maintains an appropriate systemic pH balance is poorly understood. The work described here is aimed at proving that a protein called the "soluble adenylate cyclase" can act as this elusive sensor, and that it signals another protein called a proton pump to remove acid from the body. This work, therefore, sets out to understand the mechanisms underlying a basic physiological function that is necessary for survival. We hope to identify new protein targets for the development of new therapies and strategies to correct acid base imbalances (known as acidosis or alkalosis) in the body.

描述（由申请人提供）： 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的分泌仍然是一个谜。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/CO2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。在狭窄范围内维持包括血液在内的体液（包括血液）的酸/碱（即pH）水平对正常健康和所有细胞和器官系统的功能至关重要。肾脏在此过程中通过将过量的酸或多余的碱通过排泄中的尿液中的尿液中的核心发挥作用。当前，肾脏检测并保持适当的全身pH平衡的感应机制知之甚少。此处描述的工作旨在证明一种称为“可溶性腺苷酸环化酶”的蛋白质可以充当该难以捉摸的传感器，并指示另一种称为质子泵的蛋白质以从体内去除酸。因此，这项工作旨在了解生存所必需的基本生理功能的基础机制。我们希望确定新的蛋白质靶标，以开发新的疗法和策略，以纠正体内酸碱失衡（称为酸中毒或碱中毒）。