Aquaporin Regulation: A New Translational Route to Cytotoxic CNS Oedema Therapies

水通道蛋白（AQP）控制所有组织中的水流。我们发现AQP的功能是通过调节质膜中AQP4水孔的数量来响应强压或缺氧的变化。可逆的囊泡到质膜的再定位是由机械敏感通道的激活引发的，导致细胞内Ca2+水平增加和cAMP积累，导致钙调素依赖性磷酸化和细胞骨架重组。为了推动该领域的发展，我们现在需要充分了解所涉及的机制，这是该项目的总体目标。这一新的认识将为水失衡疾病提供新的靶点，并为指导寻找新的抑制剂提供平台。我们开发了一种基于细胞表面生物素化和ELISA检测细胞表面AQP的内源性AQP质膜定位方法，目前采用24孔板形式。将其扩展到96孔和384孔格式，将使用高含量化合物文库为AQP再定位抑制剂提供高通量筛选系统。目标1：利用高含量筛选方法鉴定AQP抑制剂。我们的96孔实验测量细胞肿胀。高含量筛选技术的进步将被用于将该分析调整为基于图像的384孔泛荧光格式，量化细胞质到膜的易位和细胞表面分子的再循环。通过精心挑选的经过批准的、有注释的和多样化的化学组合进行筛选，将确定特异性调节AQP4亚细胞定位的新分子。确认的结果将进行细胞表面生物素化试验。将选择药代动力学合适的靶点进行体内概念验证试验。将使用一套细胞药理学工具深入分析优先命中，以阐明表型，蛋白质组学和转录组学水平的作用机制。目的2：明确AQP4调控的完整机制。为了获得完整的机制框架，我们将在分子细节上研究钙内流如何触发AQP4重新定位。目前未知的囊泡和调节蛋白在响应星形胶质细胞的张力变化时将AQP4转移到质膜上的分子特性将通过分子和细胞生物学工具，我们的抑制剂面板和蛋白质组学/脂质组学分析来确定。将调查AQP4同质寡聚化与贩运之间的联系。AQP4与调节其膜丰度的蛋白之间的相互作用将被表征。全长AQP4和调节性AQP4复合物的结构表征将提供一个平台，可以直接用于寻找新的抑制剂。

Aquaporins (AQP) control water flow in all tissues. We have discovered that AQP function is controlled by regulating the number of AQP4 water pores in the plasma membrane in response to changes in tonicity or hypoxia. Reversible vesicle-to-plasma-membrane relocalization is triggered by activation of mechanosensitive channels leading to increased intracellular Ca2+ levels and cAMP accumulation, resulting in calmodulin-dependent phosphorylation and cytoskeletal reorganization. To move the field forward, we now need to fully understand the mechanisms involved, which is the overall aim of this project. This new understanding will provide novel targets for diseases of water imbalance and a platform to guide the search for new inhibitors. We have developed an assay for endogenous AQP plasma membrane localization based on cell-surface biotinylation and ELISA detection of cell-surface AQP, currently in 24-well plate format. Scaling this to 96- and 384-well format will provide a high-throughput screening system for inhibitors of AQP relocalization using high content compound libraries.The objectives of this studentship are:Objective 1: Identify AQP inhibitors using high-content screening. Our 96-well assay measures cell swelling. Advances in high-content screening will be used to adapt this assay into a robust image-based 384-well transfluor format that quantifies cytoplasmic-to-membrane translocation and recycling of cell-surface molecules. Screening across a carefully-selected panel of approved, annotated and diverse chemical sets will identify new molecules that specifically modulate AQP4 sub-cellular localization. Confirmed hits will progress to our cell-surface biotinylation assay. Pharmacokinetically-appropriate hits will be selected for proof-of-concept testing in vivo. Prioritized hits will be profiled in-depth using a suite of cellular pharmacology tools to elucidate mechanism-of-action at phenotypic, proteomic and transcriptomic levels.Objective 2: Define the complete mechanism of AQP4 regulation. To obtain a complete mechanistic framework, we will examine in molecular detail how calcium influx triggers AQP4 relocalization. The currently-unknown molecular identity of vesicles and regulatory proteins transferring AQP4 to and from the plasma membrane in response to tonicity changes in astrocytes will be identified using molecular- and cell-biology tools, our inhibitor panel and proteomic/lipidomic analyses. The link between AQP4 homo-oligomerisation and trafficking will be investigated. Interactions between AQP4 and proteins that regulate its membrane abundance will be characterized. Structural characterization of full-length AQP4 and regulatory AQP4 complexes will provide a platform that can be directly exploited in the search for new inhibitors.