Trafficking of NaPilla in brush border microvilli

描述（由申请方提供）：无机磷酸盐（Pi）浓度紊乱和Pi重吸收受损是常见的临床问题。众所周知，衰老、糖尿病、恶性肿瘤、肾衰竭、酒精中毒、移植、AIDS和几种治疗药物主要通过影响肾小管Pi转运引起低磷酸盐血症或高磷酸盐血症或与之相关。肾脏在Pi稳态的调节中起着关键作用。迄今为止的证据表明，大多数肾小管Pi调节饮食和激素因素介导的Ha型肾磷酸钠协同转运蛋白（NaPilla）。这些因子通过改变协同转运蛋白插入刷状缘膜（BBM）微绒毛和从刷状缘膜（BBM）微绒毛中取回来改变NaPilla表面丰度和活性。然而，人们对NaPilla进出细胞膜的确切事件知之甚少。这些研究一直受到限制，无法可视化蛋白质运输的微绒毛与高分辨率在真实的时间。我们的实验室已经开发了一种新的应用全内反射荧光显微镜（TIR-FM），它允许可视化的运输事件在刷状缘微绒毛在真实的时间。拟议的研究将使用TIR-FM显微镜结合光漂白后荧光恢复（FRAP）和图像相关光谱（ICS）技术来研究细胞外Pi和PTH的改变如何调节BBM微绒毛中的NaPilla运输。这些研究将在对生肾（OK）细胞中进行，这是一种完善的近端小管模型。NaPilla运输也被认为是由PDZ蛋白（将协同转运蛋白连接到细胞骨架的支架蛋白）和细胞骨架本身调节的。拟议的工作将调查的NaPilla贩运BBM微绒毛的调制响应于细胞外Pi或PTH的PDZ蛋白和细胞骨架的改变。这项工作将使用动态成像技术，在真实的时间在单个蛋白质水平上研究生理过程。这些技术，除了广泛适用于其他过程，将允许洞察到肾的Pi稳态的调节，这是至关重要的正常细胞功能。这些知识可能最终导致开发新的疗法来治疗磷酸盐失衡。

DESCRIPTION (provided by applicant): Disorders of inorganic phosphate (Pi) concentration and impairment in Pi reabsorption are common clinical problems. Aging, diabetes mellitus, malignancy, renal failure, alcoholism, transplantation, AIDS, and several therapeutic drugs are well known to cause or to be associated with hypophosphatemia or hyperphosphatemia, mainly by affecting renal tubular Pi transport. The kidney plays a critical role in the regulation of Pi homeostasis. The evidence to date indicates that the majority of renal tubular Pi regulation by dietary and hormonal factors is mediated by the type Ha renal sodium phosphate cotransporter (NaPilla). These factors alter NaPilla surface abundance and thus activity by altering the cotransporter's insertion into and retrieval from brush border membrane (BBM) microvilli. Relatively little is known, however, about the precise events governing NaPilla trafficking to and from the cell membrane. These studies have been limited by the inability to visualize protein trafficking in microvilli with high resolution in real time. Our lab has developed a novel application of total internal reflection fluorescence microscopy (TIR-FM) which allows visualization of trafficking events in brush border microvilli in real time. The proposed studies will use TIR-FM microscopy coupled with fluorescence recovery after photobleaching (FRAP) and image correlation spectroscopy (ICS) techniques to examine how alterations in extracellular Pi and PTH modulate NaPilla trafficking in BBM microvilli. These studies will be performed in opposssum kidney (OK) cells, a well-established model of the proximal tubule. NaPilla trafficking is also belived to be regulated by PDZ proteins (scaffolding proteins that link the cotransporter to the cytoskeleton) and by the cytoskeleton itself. The proposed work will investigate modulation of NaPilla trafficking in BBM microvilli in response to alterations in extracellular Pi or PTH by PDZ proteins and by the cytoskeleton. This work will use dynamic imaging techniques to study physiologic processes at the single protein level in real time. These techniques, besides being widely applicable to other processes, will allow insights into the renal regulation of Pi homeostasis which is crucial for normal cellular function. This knowledge may ultimately lead to the development of novel therapies to treat phosphate imbalance.