Regulation of proton pump trafficking in kidney

描述(由申请人提供)： 间质细胞(IC)通过增加或减少肾脏集合管中的酸分泌来对血液中的pH变化作出反应。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，因为血液的pH值偏离了其正常值7.4。液泡H+ATPase(V-ATPase)是IC酸/碱平衡功能的核心，但这些细胞如何检测环境信号使它们能够适当地调节质子分泌仍然是一个谜。长期以来，人们一直认为肾脏中存在酸或碳酸氢盐传感器，但这种检测系统的身份以及这种系统如何发送信号来改变ICs的酸/碱运输机制仍有待确定。基于前一个资助时期的工作，我们在这里提出了可溶性腺苷环化酶(SAC)是最受欢迎的肾脏酸碱传感器。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，它非常适合用作IC中的碳酸氢盐/二氧化碳传感角色。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性分泌能力。我们认为，V-ATPase和SAC是一个局部信号传递过程中的合作伙伴，该过程调节V-ATPase在特定的膜微域中的靶向和运输，以调节嵌入细胞的功能和肾脏质子的分泌。我们的目标是：1)研究SAC在调节V-ATPase介导的肾上皮细胞质子分泌中的作用；2)确定V-ATPase是否与细胞骨架蛋白(肌动蛋白、明胶蛋白、Drebrin、Nadrin和Myosin VI)形成局部微复合体，调节IC中V-ATPase膜的堆积和质子的分泌。这些研究将使用多学科方法，包括独特的动物模型、分离的荧光分类嵌入细胞和细胞培养，以及包括静态和实时共聚焦显微镜在内的成像技术来跟踪V-ATPase运输。囊泡酸化、ATPase活性和质子选择性自参比微电极的测定将监测V-ATPase在内体和质膜上的功能表达。荧光(Forsman)、共振能量转移(FRET)和蛋白质-蛋白质相互作用分析将分析V-ATPase亚基在质子分泌刺激过程中是否与SAC和/或细胞骨架蛋白质相互作用。体外分析、突变分析和磷酸化蛋白质组学将探讨cAMP/PKA介导的V-ATPase磷酸化在这些相互作用中的作用。我们认为V-ATPase是一个局部化的多蛋白复合体的中心伙伴，它通过调节嵌入细胞中依赖V-ATPase的酸化机制来感知和响应普遍存在的酸碱条件。将包括血液在内的体液的酸/碱(即酸碱度)保持在一个狭窄的范围内，对正常健康和所有细胞和器官系统的功能至关重要。肾脏通过感知和排出尿液中的过量酸或过量碱，在这一过程中起着核心作用。目前，肾脏检测和维持适当的系统pH平衡的感知机制还知之甚少。这里描述的工作旨在证明，一种名为“可溶性腺苷环化酶”的蛋白质可以充当这种难以捉摸的传感器，并且它向另一种名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潜在机制。我们希望确定新的蛋白质靶点，用于开发新的治疗方法和策略，以纠正体内的酸碱失衡(称为酸中毒或碱中毒)。

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.