Regulation of proton pump trafficking in kidney

肾脏质子泵运输的调节

基本信息

批准号：
7797683
负责人：
Dennis Brown
金额：
$ 37.03万
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/7797683
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 Cell Culture Techniques Cell membrane Cell physiology Cells Complex Confocal Microscopy Cues Cyclic AMP Cytoskeletal Proteins Detection Development Disease Endosomes Energy Transfer Epithelial Cells Equilibrium Excretory function Fluorescence Fluorescence Resonance Energy Transfer 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 in vitro Assay interdisciplinary approach myosin VI nadrin protein complex protein protein interaction response second messenger sensor trafficking vacuolar H+-ATPase

项目摘要

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）：闰细胞 (IC) 通过增加或减少肾集合管中的酸分泌来响应血液 pH 值的变化。当血液 pH 值偏离正常值 7.4 时，这一过程的功能障碍会导致不同器官系统的病理生理紊乱。液泡 H+ATP 酶 (V-ATP 酶) 是 IC 酸/碱稳态功能的核心，但这些细胞如何检测环境信号以适当改变质子分泌仍然是个谜。长期以来，人们一直认为肾脏中存在酸或碳酸氢盐传感器，但该检测系统的身份以及该系统如何传输信号以修改 IC 的酸/碱传输机制仍有待确定。基于之前资助期间开展的工作，我们在此提出可溶性腺苷酸环化酶（sAC）是备受追捧的肾脏酸/碱传感器。这种蛋白质在碳酸氢根离子的直接刺激下产生第二信使 cAMP。因此，它非常适合 IC 中的碳酸氢盐/CO2 传感作用。我们假设 sAC 传感器响应酸/碱信号而产生的 cAMP 可以改变嵌入细胞的酸分泌能力。我们认为 V-ATP 酶和 sAC 是局部信号传导过程中的合作伙伴，该过程调节特定膜微域中 V-ATP 酶的靶向和运输，以调节嵌入细胞功能和肾质子分泌。我们的目标是：1) 表征 sAC 在调节肾上皮细胞 V-ATP 酶介导的质子分泌中的作用，以及 2) 确定 V-ATP 酶和细胞骨架蛋白（肌动蛋白、凝溶胶蛋白、drebrin、纳德蛋白和肌球蛋白 VI）是否形成局部微复合物，调节 IC 中 V-ATP 酶膜积累和质子分泌。这些研究将采用多学科方法，包括独特的动物模型、分离的荧光分选的嵌入细胞和细胞培养物，以及成像技术（包括静态和实时共聚焦显微镜）来跟踪 V-ATP 酶运输。囊泡酸化、ATP 酶活性和质子选择性自参考微电极的测定将监测内体和质膜中 V-ATP 酶的功能表达。荧光 (Forsman) 共振能量转移 (FRET) 和蛋白质-蛋白质相互作用测定将剖析 V-ATP 酶亚基在刺激质子分泌过程中是否与 sAC 和/或细胞骨架蛋白相互作用。体外测定、突变分析和磷酸蛋白质组学将探讨 cAMP/PKA 介导的 V-ATP 酶磷酸化在这些相互作用中的作用。我们认为 V-ATP 酶是局部多蛋白复合物的核心伙伴，通过调节嵌入细胞中 V-ATP 酶依赖性酸化机制来感知和响应普遍的酸/碱条件。将包括血液在内的体液的酸/碱（即 pH）水平维持在狭窄的范围内对于正常健康以及所有细胞和器官系统的功能至关重要。肾脏在这个过程中发挥着核心作用，通过排泄到尿液中来感知和消除过量的酸或过量的碱。目前，人们对肾脏检测和维持适当的全身 pH 平衡的传感机制知之甚少。这里描述的工作旨在证明一种称为“可溶性腺苷酸环化酶”的蛋白质可以充当这种难以捉摸的传感器，并且它向另一种称为质子泵的蛋白质发出信号，以清除体内的酸。因此，这项工作旨在了解生存所必需的基本生理功能的机制。我们希望找到新的蛋白质靶标，用于开发新疗法和纠正体内酸碱失衡（称为酸中毒或碱中毒）的策略。