Trafficking of NaPilla in brush border microvilli

描述(申请人提供)：无机磷(PI)浓度紊乱和PI重吸收障碍是常见的临床问题。众所周知，衰老、糖尿病、恶性肿瘤、肾功能衰竭、酒精中毒、移植、艾滋病和几种治疗药物可引起或与低磷血症或高磷血症相关，主要是通过影响肾小管PI转运。肾脏在PI动态平衡的调节中起着关键作用。到目前为止的证据表明，饮食和激素因素对肾小管PI的调节大部分是由Ha肾磷酸钠共转运体(NaPilla)介导的。这些因素改变了NaPilla表面的丰度，从而通过改变共转运蛋白插入和取回刷状边缘膜(BBM)微绒毛而改变了NaPilla表面的活性。然而，关于NaPilla进出细胞膜的确切事件，人们知之甚少。这些研究由于无法实时、高分辨率地可视化蛋白质在微绒毛中的运输而受到限制。我们的实验室开发了一种新的全内反射荧光显微镜(TIR-FM)的应用，它可以实时显示灌木边缘微绒毛中的运输事件。这项研究将使用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的变化如何调节BBM微绒毛中NaPilla的运输。这些研究将在奥波普苏斯肾(OK)细胞中进行，这是一种成熟的近端小管模型。NaPilla的贩运也被认为受到PDZ蛋白(将辅转运蛋白与细胞骨架联系起来的支架蛋白)和细胞骨架本身的调控。这项拟议的工作将研究PDZ蛋白和细胞骨架对细胞外PI或PTH的改变对NaPilla在BBM微绒毛中运输的调节。这项工作将使用动态成像技术来实时研究单一蛋白质水平的生理过程。这些技术除了广泛适用于其他过程外，还将使人们能够深入了解肾脏对PI稳态的调节，这对正常的细胞功能至关重要。这一知识可能最终导致治疗磷酸盐失衡的新疗法的开发。

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.