EnaC regulation in the kidney by vesicle trafficking and recycling

描述(由申请人提供)： 肾脏离子和水调节的缺陷与许多疾病有关，包括肾源性尿崩症、高血压、巴特综合征、吉特曼综合征、假性低醛固酮增多症1型和利德尔综合征。肾脏皮质集合管中的盐和水的运输是通过特定的通道进行的，这些通道受到激素反馈机制的严格控制。拟议的研究将定义和描述通过囊泡运输和再循环来调节通道的新机制。这些研究的目的是为了研究在CCD根尖膜上最常见的三种转运蛋白，即上皮钠、钾和水通道是否受小泡运输的共同定位和共同调节。利用小鼠细胞系进行的研究将确定这些贩运事件的潜在生理调节。研究将确定在这些通道的调节循环中重要的特定细胞机制。一种多学科的方法将首先通过免疫细胞化学标记以及荧光和电子显微镜成像来识别亚细胞囊泡运输间隔。接下来，将用生化方法验证通道在这些囊泡隔室中的定位。贩运事件的动态将使用活细胞成像和电生理技术来确定。最后，将阐明调节这些渠道循环的机制。通过研究Rab蛋白在受调控的通道循环中的作用，将确定参与通道运输的基本细胞成分。这些基础细胞生物学研究将进一步了解肾脏中水和离子通道的调节。这不仅有助于理解盐和水平衡所涉及的基本的动态平衡过程，更重要的是，这项工作将为这一调控中与导致病理生理疾病状态的通道调控不当相关的潜在缺陷提供见解。

DESCRIPTION (provided by applicant): Defects in ion and water regulation in the kidney have been associated with numerous diseases including, nephrogenic diabetes insipidus, hypertension, Bartter syndrome, Gitelman syndrome, pseudo-hypoaldosteronism type 1 and Liddle syndrome. Salt and water transport in the cortical collecting duct (CCD) of the kidney is mediated through specific channels which are tightly controlled by hormonal feedback mechanisms. The proposed studies will define and characterize novel mechanisms of channel regulation by vesicle trafficking and recycling. The studies aim to investigate whether three of the most common transporters at the CCD apical membranes, namely the epithelial sodium, potassium and water channels are co-localized and co-regulated by vesicle trafficking. Studies using a mouse CCD cell line will define the underlying physiological regulation of these trafficking events. Studies will identify specific cell machinery important in the regulated recycling of these channels. A multidisciplinary approach will first identify the sub-cellular vesicle trafficking compartments by immuno-cytochemical labeling and fluorescent and electron microscopic imaging. Next, the localization of channels in these vesicle compartments will be verified biochemically. The dynamics of the trafficking events will be determined using live-cell imaging and electrophysiological techniques. Finally, the mechanisms which regulate the recycling of these channels will be elucidated. By investigating the role of Rab-proteins in regulated channel recycling the essential cellular components involved in channel trafficking will be determined. These basic cell biological studies will further an understanding of water and ion channel regulation in the kidney. This will help not only to understand the essential homeostatic processes involved in salt and water balance, but more importantly, the work will provide insights into underlying defects in this regulation associated with channel misregulation which result in pathophysiological disease states.