Mechanistic Characterization of Calcium-Activated Chloride Channels in Retinal Pigment Epithelium

视网膜色素上皮中钙激活氯离子通道的机制表征

• 批准号: 10052837
• 负责人: Tingting Yang
• 金额: $ 20.12万
• 依托单位: COLUMBIA UNIVERSITY HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-05-01 至 2023-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10052837
• 关键词: Alanine; Animal Model; Anions; CRISPR/Cas technology; Calcium; Calcium Signaling; Cell Line; Cells; Chloride Channels; Clinical; DNA cassette; Degenerative Disorder; Disease; Electrooculogram; Electrophysiology (science); Engineering; Evolution; Family; Funding; Genes; Genetic; Genomics; Human; Individual; Investigation; Ion Channel; Ion Transport; Knock-out; Knockout Mice; Light; Link; Location; Mediating; Membrane Proteins; Mus; Mutagenesis; Mutation; Organ; Pathway interactions; Patients; Phenotype; Physiological; Physiology; Play; Publishing; Regulation; Research; Retina; Retinal; Retinal Degeneration; Role; Site-Directed Mutagenesis; Structural Protein; Structure; Structure of retinal pigment epithelium; Surface Properties; System; Time; Transmembrane Domain; Water; Work; X-Ray Crystallography; basolateral membrane; cell type; flexibility; genome editing; human pluripotent stem cell; inhibitor/antagonist; interdisciplinary approach; interest; knockout gene; member; mutant; patch clamp; protein structure; response; stem cell technology

Project Summary ! Ca2+-activated Cl- channels (CaCCs) open in response to increases of intracellular Ca2+ and selectively conduct Cl- and other anions. To date, two families of CaCCs, namely BESTROPHINs and TMEM16s, have been identified, among which three members- BESTROPHIN1 (BEST1), TMEM16A and TMEM16B have been proposed to function in human retinal pigment epithelium (RPE). BEST1 is predominantly expressed in RPE and genetically linked to a spectrum of retinal degenerative disorders. However, Best1 knockout mice did not display any retinal phenotype or Cl- current abnormality, arguing against the idea that BEST1 is an essential CaCC in RPE. Although human and mice may have fundamental differences on the genetic requirement of CaCCs in their RPEs, no direct evidence has been documented to support this hypothesis. TMEM16A and TMEM16B, on the other hand, are widely expressed in a variety of cell types including RPE. Their CaCC roles in RPE were suggested by studies in animal models and cell lines, but have not yet been examined in human RPE. Therefore, the physiological contributions of the three candidate CaCCs in human RPE still remain a mystery. This deficit is mainly due to the technical challenges: 1) the accessibility to native human RPE cells is very limited, and it is hard to perform gene manipulation with them; 2) currently available Cl- channel inhibitors cannot effectively distinguish BEST1, TMEM16A and TMEM16B. In this proposal, we aim to use multidisciplinary approaches, including CRISPR/Cas9-mediated genome editing, stem cell technology, whole-cell patch clamp and X-ray crystallography, to define the functional CaCC(s) in human RPE (in Aim 1), and to conduct an unprecedented mechanistic investigation on the CaCC activity and physiological role of BEST1 (in Aims 2 and 3). Our proposed work will reveal basic principles of CaCC function and regulation in human RPE, thereby making significant contributions to multiple fields of research including calcium signaling, ion transport, membrane protein structure and retinal physiology. Moreover, the pipelines established in this work can be generally applied to study ion channels and/or genes of interest in other human organs.

项目摘要 好了！ 钙激活的氯离子通道(CaCC)在细胞内钙离子和钙离子浓度升高时开放 选择性地传导氯离子和其他阴离子。到目前为止，有两个CaCC家族，即Bestrophins 和TMEM16，其中3个成员为BESTROPHIN1(BEST1)， TMEM16A和TMEM16B已被提出在人视网膜色素上皮中发挥作用 (RPE)。Best1主要在RPE中表达，并在基因上与一系列视网膜病变有关 退化性疾病。然而，Best1基因敲除小鼠没有表现出任何视网膜表型或 CL-电流异常，反对BEST1是RPE中必不可少的CACC的观点。 尽管人类和小鼠在基因需求上可能有根本的差异 尽管在他们的RPE中发现了CaCC，但没有直接证据支持这一假设。 另一方面，TMEM16A和TMEM16B在各种类型的细胞中广泛表达 包括RPE。在动物模型和细胞中的研究表明它们在RPE中的CACC作用 株，但尚未在人类RPE中进行检查。因此，生理学的贡献 人类RPE中的三个候选CaCC中的哪一个仍然是一个谜。这一逆差主要是由于 技术挑战：1)天然人类RPE细胞的可及性非常有限，而且 难以用它们进行基因操作；2)目前可用的氯通道抑制剂不能 有效区分BEST1、TMEM16A和TMEM16B。在这项提案中，我们的目标是使用 多学科方法，包括CRISPR/Cas9介导的基因组编辑、干细胞 用全细胞膜片钳和X射线结晶学技术确定功能性CACC(S) 在人类RPE中(在目标1中)，并对 BEST1的CACC活性和生理作用(目标2和3)。我们提议的工作将揭示 CACC在人RPE中作用和调节的基本原理，从而具有重要意义 对多个研究领域的贡献，包括钙信号、离子转运、膜 蛋白质结构和视网膜生理学。此外，在这项工作中建立的管道可以 通常用于研究其他人体器官中的离子通道和/或感兴趣的基因。