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

抑制肠道顶端 cAMP/cGMP 转运蛋白 (MRP4) 诱发腹泻

• 批准号: 8755665
• 负责人: Anjaparavanda P Naren
• 金额: $ 22.38万
• 依托单位: CINCINNATI CHILDRENS HOSP MED CTR
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-01-01 至 2018-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8755665
• 关键词: ATP-Binding Cassette Transporters; Agonist; Amino Acids; Apical; Biological Assay; C-terminal; Cell membrane; Cells; Chemosensitization; Chloride Ion; Chlorides; Cholera Toxin; Complex; Coupled; Coupling; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Cyclic GMP; Cyclic Nucleotides; Cystic Fibrosis Transmembrane Conductance Regulator; Diarrhea; Diffusion; Disease; Enterocytes; Epithelial Cells; Fluorescence Resonance Energy Transfer; Gastrointestinal Diseases; Gastrointestinal tract structure; Goals; Health; Health Care Costs; High Pressure Liquid Chromatography; Human; In Vitro; Individual; Intestines; Knockout Mice; Laboratories; Lateral; Lead; Length; Life; Macromolecular Complexes; Measurement; Mediating; Membrane; Methods; Microscope; Monitor; Morbidity - disease rate; Mus; Peptides; Physiological; Probenecid; RNA; Regulation; Research; Signal Pathway; Specificity; Surface; Tail; Testing; alpha Toxin; apical membrane; base; clinically relevant; crosslink; inhibitor/antagonist; knock-down; molecular assembly/self assembly; mortality; mouse model; particle; polypeptide C; protein protein interaction; research study; sensor; stoichiometry

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.

项目概要/摘要： 该提案的假设是 cAMP 转运蛋白 (MRP4) 和 CFTR Cl-通道在肠上皮细胞内物理上和功能上耦合。长期来看 该实验室的目标是 (I)、定义蛋白质-蛋白质相互作用的机制 调节CFTR Cl-通道功能和（II），致力于做出长期的努力 对了解与腹泻疾病相关的胃肠道疾病做出了贡献。具体目标 该提案的内容是： 具体目标 1. 检验 MRP4 是肠道中顶端 cAMP 转运蛋白的假设 抑制该转运蛋白会加剧霍乱毒素 (CTX) 引起的腹泻。四个子目标将 经检验，它们是(1a)。测试 MRP4 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)。测试是否抑制 MRP4 和 MRP4 沉默（使用 Si-RNA）增强心尖部 CFTR 依赖性短路电流 极化肠上皮细胞膜和切除的小鼠肠道。 (1c)。测试是否MRP4 抑制可增强小鼠霍乱毒素 (CTX) 诱导的和 CFTR 依赖性分泌性腹泻 并测试 CFTR 敲除小鼠是否无法对 CTX 和 MRP4 抑制做出反应。 (1d)。测试是否 MRP4 敲除小鼠更容易受到 CTX 诱导的分泌性腹泻的影响，因此需要测试 MRP5 是否 抑制剂不能诱导分泌。 具体目标 2. 检验 cAMP 存在物理和功能耦合的假设 肠道顶端质膜处或附近的转运蛋白 (MRP4) 和 CFTR Cl 通道 上皮细胞。将测试三个子目标，它们是（2a）。测试 cAMP 转运蛋白是否 (MRP4) 与 PDZK1 和 CFTR 形成大分子复合物，并定义其化学计量 肠上皮细胞质膜中的 CFTR:PDZK1 和 MRP4:PDZK1 复合物。 (2b)。测试 如果含有大分子复合物的 cAMP 转运蛋白的破坏会抑制 CFTR 功能 并测试 CFTR 和 MRP4 在血浆中的横向迁移率是否增加（高扩散率） 膜。 (2c)。测试 cAMP 是否在质膜处或质膜附近积聚​​（使用 膜相关的 cAMP 传感器）在抑制 cAMP 转运蛋白后。 这些研究将证明两种 ABC 转运蛋白（CFTR 和 MRP4）可以 功能上和物理上耦合，MRP4 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治疗方法和目标 某些胃肠道疾病，例如分泌性腹泻和炎症性肠病。这些研究将， 因此，对于患有某些形式的腹泻的个体具有临床意义。