Deciphering polycystin-dependent electric signaling of primary cilia in the renal system

破译肾脏系统初级纤毛的多囊蛋白依赖性电信号传导

• 批准号: 10693400
• 负责人: Kotdaji Ha
• 金额: $ 9万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-02 至 2024-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10693400
• 关键词: Affect; Autosomal Dominant Polycystic Kidney; Biological Assay; Biology; C-Type Lectins; Cations; Cell Surface Extensions; Cell membrane; Cells; Characteristics; Cilia; Clustered Regularly Interspaced Short Palindromic Repeats; Complex; Cyst; Cystic kidney; Data; Development; Disease; Electrophysiology (science); End stage renal failure; Epithelial Cells; Etiology; Foundations; Functional disorder; Glean; Goals; Human; Impairment; Incentives; Integral Membrane Protein; Ion Channel; Ions; Kidney; Kidney Diseases; Knowledge; Lead; Mammalian Cell; Measures; Membrane; Mendelian disorder; Mentors; Molecular; Mus; Mutation; Organ; Organelles; Organoids; Pathogenicity; Pathway interactions; Patients; Phase; Phenotype; Physiological; Physiology; Play; Postdoctoral Fellow; Preparation; Proteins; Publications; Publishing; Regulation; Renal tubule structure; Role; Second Messenger Systems; Signal Transduction; Specificity; Stimulus; Structure; Supervision; Surface; System; Techniques; Testing; Time; Training; Tubular formation; Universities; Variant; Washington; Work; career; ciliopathy; clinically significant; disease-causing mutation; effective therapy; experimental study; human disease; insight; molecular phenotype; mutant; patch clamp; prevent; regenerative approach; renal epithelium; tool

Project Summary/Abstract Primary cilia are unique organelles that protrude from the cell membrane of almost all vertebrate cells. Defective primary cilia, caused by mutations in proteins required for cilia formation or signaling, can result in ciliopathies, pleiotropic diseases affecting multiple organs. In particular, Autosomal Dominant Polycystic Kidney Disease (ADPKD), the most prevalent monogenic disease leading to end-stage renal disease (ESRD), is considered ciliopathy. The polycystin channel complex, formed by the transmembrane proteins PC-1 and PC-2, is abundantly expressed primary cilia, and mutations in PC-1 and PC-2 account for the vast majority of ADPKD. To date, we only have a limited functional understanding of how the polycystin complex commands the electric signaling of primary cilia via cation flux (K+, Na+, and Ca2+) due to technical challenges in characterizing channel activity of ciliary membranes. To better understand, I established ciliary patch-clamp recordings to measure the activity of endogenous polycystin channels directly from the ciliary membrane. I published in 2020 that the C- type lectin domain (CTL) of the PC-1 N-terminus plays a crucial role in polycystin activation, highlighting the indispensable participation of PC-1 subunits. In this proposal, using my established assays, I will determine how ADPKD-causing mutations within the N-terminus of PC-1 impair polycystin function. Further, I will establish ciliary patch-clamp recordings of primary cilia within kidney organoids, allowing me for the first time to functionally connect the molecular phenotype of impaired channel activity with the macroscopic phenotype of cyst formation. During the mentored K99 phase, I will characterize the functional impact of pathogenic variants within the PC-1 subunit using the ciliary patch-clamp recording. To understand the endogenous regulation of the polycystin complex, I will determine the specificity and potency for the cilia- enriched oxysterols to activate the polycystin complex in the cell or ciliary membrane. To complete this aim, I will receive further training in cilia biology in the Delling lab. In parallel, co-mentor Dr. Meyeon Park will guide me to understand the clinical significance of pathogenic mutants in PC-1. To expand knowledge of polycystin complex under more physiological conditions, I plan to measure electric signaling of primary cilia using kidney organoids during the R00 phase. Kidney organoids provide a powerful tool for understanding development and disease and finding new treatments and regenerative approaches. Characterizing ciliary ion channels in organoids will establish an essential milestone to understand the role of primary cilia in different segments of renal tubules and cyst development. During this time, the candidate will complete mentored training in the Department of Physiology in preparation for the independent R00 phase with the help of Dr. Benjamine S. Freedman at the University of Washington. Information gleaned from this work will be helpful to expand our knowledge of the fundamental role of primary cilia in renal physiology.

项目摘要/摘要 原发性纤毛是从几乎所有脊椎动物细胞的细胞膜伸出的独特细胞器。有缺陷的 由纤毛形成或信号传导所需的蛋白质突变引起的原发性纤毛可能导致纤毛病， 影响多个器官的多效性疾病。特别是常染色体显性多囊性肾脏疾病 （ADPKD）是导致末期肾脏疾病（ESRD）的最普遍的单基因疾病（ESRD） 纤毛病。由跨膜蛋白PC-1和PC-2形成的多囊蛋白通道复合物是 大量表达的原发性纤毛，PC-1和PC-2中的突变占ADPKD的绝大多数。 迄今为止，我们对多囊菌综合体如何命令电动的功能了解有限 由于表征通道的技术挑战，通过阳离子通量（K+，Na+和Ca2+）发信号传导 睫状膜的活性。为了更好地理解，我建立了睫状贴片记录以衡量 内源性多囊通道的活性直接从纤毛膜上。我在2020年出版了C- PC-1 N末端的类型凝集素结构域（CTL）在多囊激活中起着至关重要的作用，突出了 PC-1亚基必不可少的参与。在此提案中，使用我的既定测定法，我将确定 PC-1的N末端中引起ADPKD引起的突变如何损害多囊菌函数。此外，我会的 在肾脏器官中建立纤毛斑块钳记录，使我允许我参加 第一次将受损通道活性的分子表型与 囊肿形成的宏观表型。在指导的K99阶段，我将表征功能 PC-1亚基内致病变体的影响使用睫状贴片记录。理解 多囊体复合物的内源性调节，我将确定纤毛的特异性和效力 富集的氧甲醇激活细胞或纤毛膜中的多囊蛋白络合物。为了完成这个目标，我将 在戴林实验室接受进一步的纤毛生物学培训。同时，同事Meyeon Park博士将指导我 了解病原突变体在PC-1中的临床意义。 为了在更加生理条件下扩大对多囊蛋白复合物的知识，我计划测量电动 在R00期间，使用肾脏类器官对原发性纤毛的信号传导。肾脏器官提供了强大的工具 了解发展和疾病，并找到新的治疗方法和再生方法。 特征器官中的睫状离子通道将建立一个必要的里程碑，以了解 肾小管和囊肿发育不同部分的原发性纤毛。在此期间，候选人将 在生理学系的完整指导培训，为独立R00阶段做准备 在华盛顿大学的本杰明·S·弗里德曼（Benjamine S. Freedman）博士的帮助下。从这项工作中收集的信息将 有助于扩大我们对原发性纤毛在肾脏生理学中的基本作用的了解。