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: 9901624
• 负责人: Tingting Yang
• 金额: $ 32.4万
• 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/9901624
• 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 Degeneration; Role; Site-Directed Mutagenesis; Structure; Structure of retinal pigment epithelium; Surface Properties; System; Time; Transmembrane Domain; Water; Work; X-Ray Crystallography; basolateral membrane; cell type; flexibility; genetic manipulation; 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.

项目总结