Molecular Physiology of TMEM16/Anoctamin Proteins

TMEM16/Anoctamin 蛋白的分子生理学

• 批准号: 10466884
• 负责人: H. CRISS HARTZELL
• 金额: $ 34.32万
• 依托单位: EMORY UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-09-16 至 2023-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10466884
• 关键词: Action Potentials; Affect; Amino Acids; Anions; Asthma; Attention; Automobile Driving; Behavior Disorders; Binding; Binding Sites; Biophysical Process; Biophysics; Blood Pressure; Bodily secretions; Calcium; Calcium Chloride; Calcium ion; Carrier Proteins; Cations; Cell membrane; Cell physiology; Cells; Chloride Ion; Colitis; Computer Models; Coupled; Coupling; Data; Development; Disease; Drug Targeting; Electrophysiology (science); Embryo; Epithelial; Family; Foundations; Functional disorder; G-Protein-Coupled Receptors; Gastrointestinal Motility; Genes; Genetic; Goals; Head; Health; Hormone secretion; Human; Hypertension; Influentials; Inositol Phosphates; Integral Membrane Protein; Ion Channel; Ions; Knowledge; Link; Lipids; Liquid substance; Location; Lung diseases; Minor; Modeling; Molecular; Molecular Computations; Mus; Mutagenesis; Mutation; Neurons; Olfactory dysfunction; Pain; Perfusion; Pharmaceutical Preparations; Phosphatidylinositols; Phospholipase C; Phospholipids; Physiological; Physiology; Play; Potassium; Potassium Channel; Proteins; Regulation; Research; Role; Signal Pathway; Signal Transduction; Signaling Molecule; Site; Smooth Muscle; Sodium; Structure; Techniques; Testing; Therapeutic; Work; cell motility; cell type; drug development; human disease; hydrophilicity; insight; member; molecular dynamics; neuronal excitability; new therapeutic target; novel; novel therapeutics; paralogous gene; patch clamp; protein function; receptor; response; small molecule; therapeutic development; therapeutic target; three dimensional structure; voltage

Understanding the mechanisms by which small molecules are transported across cell membranes is a fundamental challenge in cell physiology. This application focuses on one family of transport proteins, the Anoctamins / TMEM16s, because they play diverse and indispensable roles in cellular physiology. The founding members of the Anoctamin (ANO) family are Ca2+-activated Cl- channels (ANO1 and ANO2). These channels are ubiquitously expressed and are intimately engaged in keeping our epithelia moist by driving the secretion of bodily fluids, controlling gut motility, facilitating the secretion of hormones, and regulating neuronal excitability and smooth muscle contractility, among other functions. Dysfunction of ANO1 has been implicated in a variety of human disease states including hypertension, colitis, asthma, and lung disease. Genetic disruption of the ANO1 gene in mice causes major developmental abnormalities, behavioral disorders, altered gastrointestinal motility, and ability to sense pain. Because ANO1 and ANO2 play such varied but essential roles in cell physiology, they represent novel targets for therapeutic drug development, but as yet ANOs as drug targets have received relatively little attention. Recently, 3-D structures of various ANOs including ANO1 have provided valuable insights into how these proteins work, but major questions remain. The long-range goal of our research is to understand the structure and function of ANO1 (TMEM16A) and ANO2 (TMEM16B). Specifically in this application, we focus on the regulation of ANO1 by the phospholipid phosphatidylinositol-(4,5)bisphosphate (PI(4,5)P2). While PI(4,5)P2 is a minor lipid in the cell membrane, it is clear that it plays a critical, but scantily understood, role in ANO1 and ANO2 function. We will use a combination of single-cell electrophysiology, directed mutagenesis, and computational molecular dynamics modeling to elucidate how the opening and closing of ANO1 is controlled by PI(4,5)P2 and calcium ions. There are 3 aims: (1) We will characterize the biophysical mechanisms of ANO1 and ANO2 regulation by PI(4,5)P2, the functional interactions between PI(4,5)P2 and calcium, and the structural requirements of phosphoinositides and inositol phosphates for channel regulation. (2) We will identify the amino acids involved in PI(4,5)P2 regulation and locate the PI(4,5)P2 binding sites in ANO1 and ANO2. Preliminary data provides strong support for the existence of 3 different PI(4,5)P2 binding sites in ANO1. (3) We will determine the functional roles for each of the 3 different PI(4,5)P2 binding sites in regulating ANO1 Ca2+ sensitivity, gating, and inactivation. A compelling reason for comparing ANO1 and ANO2 is that although these 2 proteins are 70% similar (57% identical) in sequence, ANO1 is stimulated by PI(4,5)P2 while ANO2 is inhibited. This difference provides a rich opportunity to understand how PI(4,5)P2 binding is coupled to channel function. These studies will answer pressing outstanding questions about the regulation of these channels that are crucial to human health and disease.

了解小分子在细胞膜上运输的机制是一个 细胞生理学的基本挑战。该应用集中于一种运输蛋白家族， Anoctamins / TMEM16S，因为它们在细胞生理学中起多种多样且不可或缺的作用。开国 Anoctamin（ANO）家族的成员是Ca2+激活的CL-通道（ANO1和ANO2）。这些渠道 无处不在表达，并通过推动分泌物的分泌而密切参与使我们的上皮保持湿润 体液，控制肠道运动，促进激素的分泌并调节神经元兴奋性 和平滑肌收缩力以及其他功能。 ANO1功能障碍已与某种 包括高血压，结肠炎，哮喘和肺部病在内的人类疾病状态。遗传破坏 小鼠中的ANO1基因会引起主要的发育异常，行为障碍，胃肠道改变 运动能力和感知疼痛的能力。因为ANO1和ANO2在细胞中扮演着如此多样化但重要的作用 生理学，它们代表了治疗药物开发的新型靶标，但由于药物靶标的ANOS 受到相对较少的关注。最近，包括ANO1在内的各种ANOS的3-D结构提供了 关于这些蛋白质的工作原理的宝贵见解，但仍然存在主要问题。我们研究的远程目标 是要了解ANO1（TMEM16A）和ANO2（TMEM16B）的结构和功能。特别是在这方面 应用，我们专注于通过磷脂磷脂酰肌醇 - （4,5）双磷酸的调节ANO1 （PI（4,5）P2）。虽然Pi（4,5）P2是细胞膜中的次要脂质，但很明显它起着至关重要但很少的作用 理解，在ANO1和ANO2函数中的作用。我们将使用单细胞电生理学的组合，定向 诱变和计算分子动力学建模，以阐明如何开放和关闭 ANO1由PI（4,5）P2和钙离子控制。有3个目的：（1）我们将表征生物物理 PI（4,5）P2的ANO1和ANO2调节机制，PI（4,5）P2和 钙，以及用于通道调节的磷酸肌醇和肌醇磷酸盐的结构需求。 （2）我们将确定涉及PI（4,5）P2调节的氨基酸，并在ANO1中找到PI（4,5）P2结合位点 和ano2。初步数据为存在3种不同的PI（4,5）P2结合位点的存在提供了强有力的支持 ano1。 （3）我们将确定在调节中的3种不同的PI（4,5）P2结合位点的功能作用 ANO1 Ca2+灵敏度，门控和失活。比较ANO1和ANO2的令人信服的理由是 尽管这两种蛋白质的序列是70％相似（57％相同），但ANO1被PI刺激（4,5）P2，而ANO1则是 ANO2被抑制。这种差异为了解PI（4,5）P2绑定如何与 通道功能。这些研究将回答有关这些调节的迫切问题 对人类健康和疾病至关重要的渠道。