Inhibition of an apical cAMP/cGMP transporter(MRP4)in the gut induces diarrhea

项目摘要/摘要： 这一提议的假设是cAMP转运体(MRP4)和 CFTRCl-通道在肠道上皮细胞内存在物理和功能上的偶联。长期的 本实验室的目标是：(I)确定蛋白质-蛋白质相互作用的机制 规范CFTRCl-通道功能和(Ii)，致力于使我们的 在了解与腹泻病相关的胃肠疾病方面的贡献。具体目标 这项建议的内容如下： 具体目标1.检验MRP4是肠道和胃肠道顶端cAMP转运体的假设 抑制这一转运蛋白可增强霍乱毒素(CTX)引起的腹泻。四个子目标将 接受测试，它们是(1a)。为了测试MRP4是否是肠上皮细胞的顶端cAMP转运体，以及 用高效液相色谱表征环核苷酸转运。(1B)。以测试MRP4抑制和 MRP4沉默(使用Si-RNA)增加心尖部CFTR依赖的短路电流 极化的肠上皮细胞膜和小鼠离体肠。(1C)。测试MRP4是否 抑制能增强霍乱毒素(CTX)诱导的小鼠分泌性腹泻和CFTR依赖 并测试cftr基因敲除小鼠是否对CTX和MRP4抑制没有反应。(1D)。来测试一下 MRP4基因敲除小鼠对CTX诱导的分泌性腹泻更敏感，并测试MRP5基因敲除小鼠是否 抑制剂不能诱导分泌。 具体目标2.检验cAMP存在生理和功能偶联的假设 肠道顶端质膜上或其附近的转运蛋白(MRP4)和CFTRCl-通道 上皮细胞。将测试三个分目标，它们是(2a)。测试营地的运输机是否 (MRP4)是与PDZK1和CFTR形成的大分子络合物，并定义了 CFTR：PDZK1和MRP4：PDZK1复合体位于肠上皮细胞质膜。(2B)。为了测试 如果含有大分子复合体的cAMP转运体的破坏抑制了CFTR功能 并测试CFTR和MRP4在血浆中的横向迁移率是否增加(高扩散速率) 薄膜。(2C)。要测试cAMP是否在质膜或质膜附近积累(使用 膜相关cAMP传感器)。 这些研究将证明两个ABC转运蛋白(CFTR和MRP4)可以是 MRP4的抑制可以增强CFTR转运蛋白的功能。 这些研究的结果将为我们提供可能的替代治疗方法和目标。 某些胃肠道疾病，如分泌性腹泻和IBD。这些研究将会， 因此，对患有某些形式腹泻的个体具有临床相关性。

PROJECT SUMMARY/ABSTRACT: The hypothesis of this proposal is that the functional activities of the cAMP transporter (MRP4) and CFTR Cl- channel are physically and functionally coupled within the gut epithelial cells. The long-term objectives of this laboratory are (I), To define the mechanism of how protein-protein interactions regulate CFTR Cl- channel function and (II), To dedicate our efforts toward making a long-term contribution in understanding gastrointestinal disorders related to diarrheal diseases. The specific aims of this proposal are: Specific Aim 1. To test the hypothesis that MRP4 is an apical cAMP transporter in the gut and inhibition of this transporter potentiates cholera toxin (CTX) induced diarrhea. Four subaims will be tested, they are (1a). To test whether MRP4 is an apical cAMP transporter in gut epithelial cells and to characterize the cyclic nucleotide transport using HPLC. (1b). To test whether MRP4 inhibition and MRP4 silencing (using Si-RNA) augments CFTR-dependent short circuit currents in the apical membrane of polarized gut epithelial cells and in excised mouse intestine. (1c). To test whether MRP4 inhibition can potentiate cholera toxin (CTX)-induced and CFTR-dependent secretory diarrhea in mice and to test if CFTR knock out mice fail to respond to CTX and MRP4 inhibition. (1d). To test whether MRP4 knock out mice are more susceptible to CTX-induced secretory diarrhea and to test if MRP5 inhibitors fail to induce secretion. Specific Aim 2. To test the hypothesis that there is a physical and functional coupling of cAMP transporter (MRP4) and CFTR Cl- channel at or near the apical plasma membrane of gut epithelial cells. Three subaims will be tested, they are (2a). To test whether the cAMP transporter (MRP4) is in a macromolecular complex with PDZK1 and CFTR and to define the stoichiometries of CFTR:PDZK1 and MRP4:PDZK1 complex in the plasma membrane of gut epithelial cells. (2b). To test if the disruption of the cAMP transporter containing macromolecular complex inhibits CFTR function and to test if the lateral mobility of CFTR and MRP4 increases (high diffusion rates) at the plasma membrane. (2c). To test whether cAMP accumulates at or near the plasma membrane (using a membrane associated cAMP sensor) upon inhibition of the cAMP transporter. These studies will demonstrate that the two ABC transporters (CFTR and MRP4) can be functionally and physically coupled and that MRP4 inhibition can augment CFTR transporter function. The results of these studies will provide us with possible alternative methods and targets for treating certain diseases of the gastrointestinal tract such as secretory diarrhea and IBD. These studies will, therefore, have clinical relevance in individuals suffering from certain forms of diarrhea.