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.

了解小分子通过细胞膜转运的机制是一个 细胞生理学的基本挑战。本申请集中于转运蛋白的一个家族， Anoctamins /TMEM 16，因为它们在细胞生理学中发挥着不同且不可或缺的作用。成立 Anoctamin（ANO）家族的成员是Ca 2+激活的Cl-通道（ANO 1和ANO 2）。这些通道 是普遍表达的，并密切参与保持我们的上皮细胞湿润，通过驱动分泌 体液，控制肠道运动，促进激素分泌，调节神经元兴奋性 和平滑肌收缩性等功能。ANO 1的功能障碍与多种 包括高血压、结肠炎、哮喘和肺病的人类疾病状态。基因破坏 ANO 1基因在小鼠中引起主要发育异常、行为障碍、胃肠道改变 能动性和痛觉由于ANO 1和ANO 2在细胞中发挥着多种多样但必不可少的作用， 虽然在生理学上，它们代表了治疗药物开发的新靶点，但作为药物靶点， 受到的关注相对较少。近来，已经提供了包括ANO 1的各种ANO的3-D结构， 这些蛋白质如何工作的宝贵见解，但主要问题仍然存在。我们研究的长期目标是 了解ANO 1（TMEM 16 A）和ANO 2（TMEM 16 B）的结构和功能。详见本 应用，我们专注于调节ANO 1的磷脂酰肌醇-（4，5）二磷酸 (PI(4（5）P2）。虽然PI（4，5）P2是细胞膜中的次要脂质，但很明显，它在细胞膜中起着关键的但很少的作用。 理解，在ANO 1和ANO 2功能中的作用。我们将使用单细胞电生理学的组合， 诱变，和计算分子动力学建模，以阐明如何打开和关闭的 ANO 1受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调节ANO 1和ANO 2的机制，PI（4，5）P2和 钙，以及磷酸肌醇和肌醇磷酸对通道调节的结构要求。 (2)我们将鉴定参与PI（4，5）P2调节的氨基酸，并定位ANO 1中的PI（4，5）P2结合位点。 ANO2。初步数据提供了强有力的支持，3个不同的PI（4，5）P2结合位点的存在， ANO1. (3)我们将确定3个不同的PI（4，5）P2结合位点中的每一个在调节细胞凋亡中的功能作用。 ANO 1 Ca 2+敏感性、门控和失活。比较ANO 1和ANO 2的一个令人信服的原因是， 尽管这两种蛋白质在序列上70%相似（57%相同），但ANO 1被PI（4，5）P2刺激， ANO 2被抑制。这种差异提供了丰富的机会来了解PI（4，5）P2结合是如何偶联到 通道功能这些研究将回答关于这些监管的紧迫的悬而未决的问题， 对人类健康和疾病至关重要的渠道。