EnaC regulation in the kidney by vesicle trafficking and recycling

描述（由申请人提供）： 肾脏中离子和水调节的缺陷与许多疾病相关，包括肾源性尿崩症、高血压、Bartter综合征、Gitelman综合征、1型假性醛固酮减少症和Liddle综合征。肾脏皮质集合管（CCD）中的盐和水运输通过特定通道介导，这些通道由激素反馈机制严格控制。拟议中的研究将定义和表征新的机制，通道调节囊泡运输和回收。这些研究的目的是调查是否在CCD顶端膜，即上皮钠，钾和水通道的三个最常见的转运共定位和共调节囊泡运输。使用小鼠CCD细胞系的研究将定义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再循环中重要的特定细胞机制。一个多学科的方法将首先确定的亚细胞囊泡运输舱免疫细胞化学标记和荧光和电子显微镜成像。接下来，将通过生物化学方法验证这些囊泡隔室中通道的定位。将使用活细胞成像和电生理技术确定贩运活动的动态。最后，将阐明调节这些通道的再循环的机制。通过研究Rab蛋白在调节通道再循环中的作用，将确定参与通道运输的基本细胞组分。这些基本的细胞生物学研究将进一步了解肾脏中的水和离子通道调节。这将不仅有助于了解盐和水平衡中涉及的基本稳态过程，更重要的是，这项工作将深入了解与导致病理生理疾病状态的通道失调相关的这种调节中的潜在缺陷。

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.