REGULATION OF NA TRANSPORT FUNCTION BY CFTR IN THYROID EPITHELIAL CELLS

CFTR对甲状腺上皮细胞NA转运功能的调节

• 批准号: 7959795
• 负责人: PEYING FONG
• 金额: $ 17.52万
• 依托单位: KANSAS STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-07-01 至 2010-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7959795
• 关键词: Address; Apical; Binding; Biological Assay; Biotinylation; Cell membrane; Cell surface; Cells; Chloride Channels; Complex; Computer Retrieval of Information on Scientific Projects Database; Cyclic AMP; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Disease; Endocytosis; Epithelial; Epithelial Cells; Family member; Family suidae; Fluorescence; Funding; Future; Grant; Hand; Health; Institution; Knowledge; Measurement; Membrane; Methodology; Microscopic; Organ; Polycystic Kidney Diseases; Preparation; Property; Proteins; Regulation; Research; Research Personnel; Resources; Scaffolding Protein; Signal Transduction; Sodium; Sodium Channel; Source; Syndrome; Thyroid Gland; United States National Institutes of Health; absorption; beta-adrenergic receptor; epithelial Na+ channel; human disease; insight; novel; tool; uptake

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The regulation of sodium absorption by epithelial cells is key to understanding the pathological disruptions in many human diseases, yet it remains poorly understood. Sodium absorption via the epithelial sodium channel, ENaC, is believed to be regulated by the Cystic Fibrosis Transmembrane Conductance Regulator, CFTR. CFTR is a multifunctional protein that not only functions as a cAMP-gated chloride channel, but that also exists in a macromolecular signalling complex, interacting directly with scaffolding proteins, upstream modulators and downstream effectors (egs. the beta-adrenergic receptor and SLC26 transporter family members). On the other hand, direct molecular interactions between CFTR and ENaC have not been demonstrated. In general, the regulation of ENaC's net contribution to epithelial transport may be accomplished by changing its gating, its single channel conductance and/or the number of functional channels in the plasma membrane. The thyroid gland is an organ in which CFTR- transporter interactions at the level of the macromolecular signalling complex may influence ENaC activity critically. The disruption of those interactions may dysregulate ENaC and cause disease. The present proposal asks whether CFTR regulates epithelial sodium absorption by influencing a constitutive property of all cells, endocytosis, and hence changing the numbers of ENaC in the membrane. With regard to mechanism, + Does activation of CFTR normally cause cells to release ATP into the apical compartment? + Does ATP stimulate endocytosis? + How does endocytosis modulate net epithelial sodium absorption? To address this hypothesis, the proposed study will use primary porcine thyroid epithelial cultures, a robust, bidirectionally transporting epithelial preparation. The approach incorporates measurements of short-circuit current and employs specific pharmacologic tools in these measurements, as well as in fluorescence microscopic assays of endocytic uptake, cell surface biotinylation, and immunolocalization studies. The combination of several methodologies will enable clear answers to the posed questions to be drawn, leading to novel insights that in turn will clear a path toward future questions. It is envisaged that these studies will fill a gap in knowledge that presently impedes overall progress in understanding diseases as diverse as Pendred syndrome, cystic fibrosis and polycystic kidney disease.

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 上皮细胞对钠吸收的调节是理解许多人类疾病的病理破坏的关键，但它仍然知之甚少。通过上皮钠通道(ENaC)的钠吸收被认为是由囊性纤维化跨膜电导调节器(CFTR)调节的。Cftr是一种多功能蛋白质，它不仅作为cAMP门控的氯离子通道发挥作用，而且还存在于大分子信号复合体中，直接与支架蛋白、上游调节器和下游效应器(EGs)相互作用。β-肾上腺素能受体和SLC26转运蛋白家族成员)。另一方面，CFTR和ENaC之间的直接分子相互作用还没有被证明。一般来说，ENaC对上皮转运的净贡献的调节可以通过改变其门控、其单通道电导和/或质膜中功能通道的数量来完成。甲状腺是一个大分子信号复合体水平上的CFTR-转运蛋白相互作用的器官，它可能对ENaC的活性产生重要影响。这些相互作用的破坏可能会扰乱ENaC并导致疾病。目前的建议是问CFTR是否通过影响所有细胞的组成属性，即内吞作用，从而改变膜上ENaC的数量来调节上皮钠的吸收。在机制方面， +cftr的激活是否通常会导致细胞将ATP释放到心尖室？ +ATP刺激内吞作用吗？ +内吞作用如何调节上皮钠的净吸收？ 为了解决这一假设，拟议的研究将使用原代猪甲状腺上皮细胞培养，一种强大的，双向运输上皮制剂。该方法结合了短路电流的测量，并在这些测量中使用了特定的药理学工具，以及在细胞内摄取、细胞表面生物素化和免疫定位研究的荧光显微镜分析中。几种方法的结合将使所提出的问题得到明确的答案，从而产生新的见解，进而为未来的问题扫清道路。预计这些研究将填补目前阻碍理解各种疾病的知识空白，如Pendred综合征、囊性纤维化和多囊肾病。