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: 7720927
• 负责人: PEYING FONG
• 金额: $ 16.46万
• 依托单位: KANSAS STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-07-01 至 2009-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7720927
• 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; Disruption; Endocytosis; Epithelial; Epithelial Cells; Family member; Family suidae; Fluorescence; Funding; Future; Grant; Hand; Institution; Knowledge; Measurement; Membrane; Methodology; Microscopic; Numbers; 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.

该子项目是利用该技术的众多研究子项目之一 资源由 NIH/NCRR 资助的中心拨款提供。子项目及 研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金， 因此可以在其他 CRISP 条目中表示。列出的机构是 对于中心来说，它不一定是研究者的机构。 上皮细胞对钠吸收的调节是了解许多人类疾病的病理破坏的关键，但人们对此仍知之甚少。据信，通过上皮钠通道 ENaC 的钠吸收受到囊性纤维化跨膜电导调节器 CFTR 的调节。 CFTR 是一种多功能蛋白，不仅具有 cAMP 门控氯离子通道的功能，而且还存在于大分子信号复合物中，与支架蛋白、上游调节剂和下游效应器（例如 β-肾上腺素能受体和 SLC26 转运蛋白家族）直接相互作用成员）。另一方面，CFTR 和 ENaC 之间的直接分子相互作用尚未得到证实。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实现。甲状腺是一个大分子信号复合物水平的 CFTR-转运蛋白相互作用可能严重影响 ENaC 活性的器官。这些相互作用的破坏可能会使 ENaC 失调并导致疾病。目前的提案询问 CFTR 是否通过影响所有细胞的组成特性（内吞作用）来调节上皮钠吸收，从而改变膜中 ENaC 的数量。从机制上来说， + CFTR 的激活通常会导致细胞将 ATP 释放到顶端室吗？ + ATP 是否刺激内吞作用？ + 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培养物，这是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，并在这些测量以及内吞摄取的荧光显微镜测定、细胞表面生物素化和免疫定位研究中采用特定的药理学工具。几种方法的结合将使所提出的问题能够得到清晰的答案，从而产生新颖的见解，从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彭德雷综合征、囊性纤维化和多囊肾病等多种疾病的整体进展的知识空白。