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: 10374118
• 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 2024-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10374118
• 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; 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 ! Ca 2+激活的Cl-通道（CaCCs）响应于细胞内Ca 2+和Ca 2+浓度的增加而开放。 选择性地传导Cl-和其它阴离子。迄今为止，两个CaCC家族，即BESTROPHIN， 和TMEM 16，已被鉴定，其中三个成员-BESTROPHIN 1（BEST 1）， 已经提出TMEM 16 A和TMEM 16 B在人视网膜色素上皮中起作用 （RPE）。BEST 1主要在RPE中表达，并与视网膜色素上皮细胞的一系列基因相关。 退行性疾病然而，Best 1基因敲除小鼠没有显示任何视网膜表型， Cl-电流异常，反对BEST 1是RPE中必需的CaCC的观点。 虽然人类和小鼠在遗传需求上可能存在根本差异， CaCC在他们的RPE中，没有直接的证据支持这一假设。 另一方面，TMEM 16 A和TMEM 16 B在多种细胞类型中广泛表达 包括RPE。在动物模型和细胞培养中的研究表明了它们在RPE中的作用。 线，但尚未在人类RPE中进行检查。因此，生理贡献 在人类RPE中的三种候选CaCC中的哪一种仍然是一个谜。这一赤字主要是由于 技术挑战：1）对天然人RPE细胞的可及性非常有限， 很难用它们进行基因操作; 2）目前可用的Cl-通道抑制剂不能 有效区分BEST 1、TMEM 16 A和TMEM 16 B。在本提案中，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 技术，全细胞膜片钳和X射线晶体学，以确定功能性CaCC（s） 在人类RPE（目标1），并进行前所未有的机制研究， BEST 1的CaCC活性和生理作用（目的2和3）。我们的工作将揭示 CaCC在人类RPE中的功能和调节的基本原理，从而使 对多个研究领域的贡献，包括钙信号传导，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。

项目成果

Structure and Function of the Bestrophin family of calcium-activated chloride channels.

• DOI: 10.1080/19336950.2021.1981625
• 发表时间: 2021-12
• 期刊: Channels (Austin, Tex.)
• 作者: Owji AP;Kittredge A;Zhang Y;Yang T
• 通讯作者: Yang T

ATP activates bestrophin ion channels through direct interaction.

ATP 通过直接相互作用激活卵黄蛋白离子通道

• DOI: 10.1038/s41467-018-05616-4
• 发表时间: 2018-08-07
• 期刊: Nature communications
• 影响因子: 16.6
• 作者: Zhang Y;Kittredge A;Ward N;Ji C;Chen S;Yang T
• 通讯作者: Yang T

Evaluating BEST1 mutations in pluripotent stem cell-derived retinal pigment epithelial cells.

• DOI: 10.1016/bs.mie.2021.01.004
• 发表时间: 2021
• 期刊: Methods in enzymology

Bestrophin-2 and glutamine synthetase form a complex for glutamate release.

• DOI: 10.1038/s41586-022-05373-x
• 发表时间: 2022-11
• 期刊: NATURE
• 影响因子: 64.8
• 作者: Owji, Aaron P.;Yu, Kuai;Kittredge, Alec;Wang, Jiali;Zhang, Yu;Yang, Tingting
• 通讯作者: Yang, Tingting

Structures and gating mechanisms of human bestrophin anion channels.

• DOI: 10.1038/s41467-022-31437-7
• 发表时间: 2022-07-04
• 期刊: Nature communications
• 影响因子: 16.6