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

项目总结/摘要： 这一提议的假设是cAMP转运蛋白（MRP 4）的功能活性和 CFTR Cl-通道在肠上皮细胞内物理上和功能上偶联。长期 本实验室的目标是（I），确定蛋白质-蛋白质相互作用的机制， 调节CFTR Cl-通道功能和（II），致力于使我们的努力长期 有助于了解与胃肠疾病相关的胃肠疾病。具体目标 该建议包括： 具体目标1。为了检验MRP 4是肠道中顶端cAMP转运蛋白的假设， 这种转运蛋白的抑制增强了霍乱毒素（CTX）诱导的腹泻。四个分目标将 测试，他们是（1A）。为了测试MRP 4是否是肠上皮细胞中的顶端cAMP转运蛋白， 使用HPLC表征环核苷酸转运。（1b）。为了测试MRP 4抑制和 MRP 4沉默（使用Si-RNA）增加了心尖部CFTR依赖性短路电流 极化肠上皮细胞膜和切除的小鼠肠。（1c）。为了测试MRP 4是否 抑制可增强霍乱毒素（CTX）诱导和CFTR依赖性分泌性腹泻小鼠 并测试CFTR敲除小鼠是否不能对CTX和MRP 4抑制反应。（1d）.来测试是否 MRP 4基因敲除小鼠对CTX诱导的分泌性腹泻更敏感， 抑制剂不能诱导分泌。 具体目标2。为了检验cAMP存在物理和功能偶联的假设， 转运蛋白（MRP 4）和CFTR Cl-通道位于或接近肠顶端质膜 上皮细胞将测试三个子目标，它们是（2a）。为了测试cAMP转运蛋白 （MRP 4）与PDZK 1和CFTR在大分子复合物中，并定义了MRP 4的化学计量。 CFTR：PDZK 1和MRP 4：PDZK 1复合物在肠上皮细胞的质膜中。（2b）。测试 如果破坏含cAMP转运蛋白的大分子复合物抑制CFTR功能， 并测试CFTR和MRP 4的横向迁移率是否在等离子体中增加（高扩散速率 膜的（2c）。为了测试cAMP是否在质膜或质膜附近积聚（使用 膜相关的cAMP传感器）。 这些研究将证明两种ABC转运蛋白（CFTR和MRP 4）可以被 功能上和物理上耦合，并且MRP 4抑制可以增强CFTR转运蛋白功能。 这些研究的结果将为我们提供可能的替代方法和治疗目标 某些胃肠道疾病，如分泌性腹泻和IBD。这些研究将， 因此，对于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.