Functional Relevance of CFTR Trafficking in Vivo to Intestinal Disease

CFTR 体内贩运与肠道疾病的功能相关性

• 批准号: 8316450
• 负责人: Nadia A. Ameen
• 金额: $ 5.32万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-04-01 至 2013-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8316450
• 关键词: Acids; Actins; Acute; Agonist; Animal Model; Apical; Bicarbonates; Binding; Binding Sites; Biochemical; Biological; Biological Assay; Cell membrane; Cell model; Cell surface; Cells; Chloride Channels; Chloride Ion; Chlorides; Cholera Toxin; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Cyclic GMP; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Diarrhea; Disease; Duodenum; Electrophysiology (science); Endocytosis; Enterocytes; Enterotoxins; Exocytosis; Fluids and Secretions; Functional disorder; Goals; Health; Heating; Immunoprecipitation; Intestinal Diseases; Intestines; Investigation; Lead; Link; Mediating; Membrane; Motor; Movement; Mus; Mutation; Organelles; Pathogenesis; Pathway interactions; Perfusion; Phosphorylation; Physiological; Physiology; RNA Interference; Rattus; Regulation; Role; Second Messenger Systems; Signal Transduction; Site-Directed Mutagenesis; Small Interfering RNA; Specificity; Surface; Tissues; Transgenic Mice; Transgenic Organisms; Vesicle; apical membrane; base; in vivo; in vivo Model; insight; jejunum; meetings; mutant; myosin VI; second messenger; trafficking

DESCRIPTION (provided by applicant): In cystic fibrosis (CF), mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel are associated with defective cAMP and acid-stimulated duodenal bicarbonate secretion, and defective cAMP and cGMP-activated fluid secretion in the intestine. On the other hand, increased activation of CFTR by cAMP and cGMP agonists elicit massive fluid secretion that result in secretory diarrhea. But the mechanisms responsible for increased or decreased activation of CFTR on the apical plasma membrane under physiological conditions in the intestine are unknown. Protein kinase A and G (PKA, PKG) phosphorylation is accepted as central to CFTR regulation. But this dogma assumes that CFTR is confined to the apical plasma membrane. We hypothesize that under physiological conditions in the intetsine, cell and region specific trafficking regulate the number and function of CFTR channels on the cell surface and contribute to disease pathogenesis. This hypothesis is supported by the following observations: (1) CFTR expression is cell and region-specific in the intestine (2) more than 50% of CFTR is in intracellular organelles in native enterocytes and supports constituitive and regulated trafficking in vivo (3) PKA and PKG regulate CFTR trafficking to the cell surface and fluid secretion in a cell and region specific manner in the intestine (4) in the duodenum, cAMP and luminal acid regulate CFTR trafficking and bicarbonate secretion (5) the endocytosis of CFTR from the apical plasma membrane in the intestine of transgenic mice lacking expression of the actin-binding motor myosin VI (Myo6 (sv/sv))is markedly defective, leads to its accumulation on the cell surface and increased CFTR-activated fluid secretion elicited by the cGMP- agonist Heat Stable Enterotoxin in vivo. Over the next five years, we propose to expand these observations and further elucidate the biological relevance of region and cell-specificity of CFTR trafficking, the role of luminal acid in regulating CFTR trafficking and function in the duodenum and the mechanism by which myosin VI regulates CFTR trafficking. We plan an integrative approach that incorporates state of the art cell biological structural, biochemical, and immunological studies including RNA silencing and in vivo perfusion and electrophysiology. These approaches, in conjunction with in vivo models of rat, and transgenic Myo6 (sv/sv) mouse intestine, polarized secretory and absorptive intestinal cells expressing endogenous CFTR and WT (wild type), DF508 and N287Y mutant CFTR-expressing cells will facilitate a comprehensive investigation to meet the defined goals of this proposal. PUBLIC HEALTH RELEVANCE In cystic fibrosis (CF), the cystic fibrosis transmembrane conductance regulator CFTR chloride channel fails to reach the plasma membrane of cells and in secretory diarrhea, CFTR function on the membrane of intestinal cells is increased. But the mechanisms that lead to CF or secretory diarrhea are unknown. This proposal seeks to understand the mechanisms that regulate CFTR movement into to the membrane under physiological conditions in the intestine in order to elucidate how dysfunction of CFTR leads to either CF or secretory diarrhea.

描述（由申请人提供）：在囊性纤维化（CF）中，囊性纤维化跨膜电导调节剂（CFTR）氯化物通道中的突变与cAMP和酸刺激的双核酸分泌和酸刺激的十二指肠碳酸氢盐分泌，以及无缺的cAMP和CGMP cAMP和CGMP激活的流体分泌。另一方面，CAMP和CGMP激动剂对CFTR的激活增加引起大规模的液体分泌，从而导致分泌性腹泻。但是，在肠道生理条件下，导致CFTR激活增加或减少的机制尚不清楚。蛋白激酶A和G（PKA，PKG）磷酸化被接受为CFTR调节的核心。但是该教条假设CFTR仅限于顶端质膜。我们假设在胸他，细胞和区域特定运输的生理条件下，调节CFTR通道在细胞表面的数量和功能，并导致疾病发病机理。 This hypothesis is supported by the following observations: (1) CFTR expression is cell and region-specific in the intestine (2) more than 50% of CFTR is in intracellular organelles in native enterocytes and supports constituitive and regulated trafficking in vivo (3) PKA and PKG regulate CFTR trafficking to the cell surface and fluid secretion in a cell and region specific manner in the intestine （4）在十二指肠和腔内酸中，调节CFTR的运输和碳酸氢盐分泌（5）在无法表达肌动蛋白结合运动肌蛋白肌醇VI（MyO6（SV/SV）的cFTR中，CFTR的CFTR的CFTR中的CFTR中的CFTR中的CFTR中的CFTR病细胞和cftress的cftressis cftress cftr有明显的cFTRE（SV/SV），并累积了CORTS的cORTS，它是cftrane coftrane coftrane coftr。 CGMP-Agonist热稳定肠毒素在体内引起的液体分泌。在接下来的五年中，我们建议扩大这些观察结果，并进一步阐明CFTR运输区域和细胞特异性的生物学相关性，Luminal Acid在调节DuodoDeNum中调节CFTR运输和功能方面的作用以及肌球蛋白VI调节CFTR的机制。我们计划了一种综合方法，该方法结合了艺术细胞生物学结构，生化和免疫学研究，包括RNA沉默和体内灌注和电生理学。 These approaches, in conjunction with in vivo models of rat, and transgenic Myo6 (sv/sv) mouse intestine, polarized secretory and absorptive intestinal cells expressing endogenous CFTR and WT (wild type), DF508 and N287Y mutant CFTR-expressing cells will facilitate a comprehensive investigation to meet the defined goals of this proposal.公共卫生在囊性纤维化（CF）中，囊性纤维化跨膜电导调节剂CFTR氯化物通道无法达到细胞的质膜和分泌腹泻，CFTR在肠细胞膜上的CFTR功能增加了。但是导致CF或分泌性腹泻的机制尚不清楚。该提案试图了解在肠道中的生理条件下将CFTR运动转化为膜的机制，以阐明CFTR功能障碍如何导致CF或分泌性腹泻。