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.

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 上皮细胞对钠吸收的调节是理解许多人类疾病中病理破坏的关键，但对它的了解仍然很少。通过上皮钠通道ENaC的钠吸收被认为是由囊性纤维化跨膜传导调节因子CFTR调节的。CFTR是一种多功能蛋白质，其不仅作为cAMP门控氯离子通道起作用，而且还存在于大分子信号传导复合物中，与支架蛋白、上游调节剂和下游效应物直接相互作用（例如，β-肾上腺素能受体和SLC 26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尚未得到证实。通常，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。甲状腺是一个器官，其中CFTR-转运蛋白在大分子信号传导复合物水平上的相互作用可能严重影响ENaC活性。这些相互作用的破坏可能会使ENaC失调并导致疾病。目前的建议是问CFTR是否通过影响所有细胞的组成特性，内吞作用，从而改变膜中ENaC的数量来调节上皮细胞的钠吸收。关于机制， + CFTR的激活是否正常导致细胞释放ATP进入顶室？ + ATP刺激内吞作用吗？ + 内吞作用如何调节上皮细胞钠的净吸收？ 为了解决这一假设，拟定的研究将使用原代猪甲状腺上皮培养物，这是一种稳健的双向转运上皮制备物。该方法结合了短路电流的测量，并在这些测量中采用特定的药理学工具，以及在荧光显微镜测定的内吞摄取，细胞表面生物素化和免疫定位研究。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，反过来又将为未来的问题扫清道路。据设想，这些研究将填补目前阻碍了解Pendred综合征，囊性纤维化和多囊肾病等各种疾病的整体进展的知识空白。