Regulation of proton pump trafficking in kidney

嵌入细胞（IC）通过增加或减少肾脏集合管中的酸分泌来响应血液中的pH变化。这一过程的功能障碍导致不同器官系统的病理生理紊乱，因为血液的pH值偏离其正常值7.4。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，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传递信号以改变IC的酸/碱转运机制仍有待确定。基于在前一个资助期进行的工作，我们在这里提出，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，它非常适合于IC中的碳酸氢盐/CO{2}传感作用。我们推测，cAMP产生的sAC传感器响应酸/碱线索可以修改的酸分泌能力的嵌入细胞。我们认为V-ATP酶和sAC是局部信号传导过程中的伙伴，该信号传导过程调节特定膜微区中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在肾上皮细胞调节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、dreplatin、nadrin和肌球蛋白VI）是否形成调节IC中V-ATP酶膜积累和质子分泌的局部微复合物。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，以跟踪V-ATP酶的运输。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V-ATP酶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-蛋白质相互作用测定将剖析在刺激质子分泌期间V-ATP酶亚基是否与sAC和/或细胞骨架蛋白相互作用。体外试验、突变分析和磷酸蛋白质组学将阐明cAMP/PKA介导的V-ATP酶磷酸化在这些相互作用中的作用。我们提出，V-ATP酶是一个中央合作伙伴在本地化的，多蛋白质复合物，传感器和响应流行的酸/碱条件下，通过调节V-ATP酶依赖的酸化机制在插入细胞。

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.