Molecular Physiology of TMEM16/Anoctamin Proteins

TMEM16/Anoctamin 蛋白的分子生理学

• 批准号: 10245101
• 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/10245101
• 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.

了解小分子跨细胞膜运输的机制是一个重要的课题 细胞生理学的根本挑战。该应用重点关注一个转运蛋白家族，即 Anoctamins / TMEM16s，因为它们在细胞生理学中发挥着多样化且不可或缺的作用。成立 Anoctamin (ANO) 家族的成员是 Ca2+ 激活的 Cl- 通道（ANO1 和 ANO2）。这些渠道 普遍表达，并通过驱动分泌来密切参与保持上皮细胞湿润 体液，控制肠道蠕动，促进激素分泌，调节神经元兴奋性 和平滑肌收缩力等功能。 ANO1 的功能障碍与多种疾病有关 人类疾病状态包括高血压、结肠炎、哮喘和肺病。基因破坏 小鼠中的 ANO1 基因导致严重的发育异常、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 在细胞中发挥着多种多样但至关重要的作用 生理学上，它们代表了治疗药物开发的新靶点，但迄今为止，ANO 作为药物靶点已经 受到的关注相对较少。最近，包括 ANO1 在内的各种 ANO 的 3D 结构提供了 关于这些蛋白质如何发挥作用的宝贵见解，但主要问题仍然存在。我们研究的长期目标 就是要了解ANO1（TMEM16A）和ANO2（TMEM16B）的结构和功能。具体在这方面 在应用中，我们重点研究磷脂酰肌醇-(4,5)二磷酸对ANO1的调节 (PI(4,5)P2)。虽然 PI(4,5)P2 是细胞膜中的一种次要脂质，但很明显它发挥着关键但很少的作用。 据了解，ANO1 和 ANO2 功能中的作用。我们将结合单细胞电生理学，定向 诱变和计算分子动力学建模，以阐明如何打开和关闭 ANO1 受 PI(4,5)P2 和钙离子控制。有 3 个目标：（1）我们将表征生物物理 PI(4,5)P2 调节 ANO1 和 ANO2 的机制，PI(4,5)P2 和 钙，以及磷酸肌醇和磷酸肌醇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ANO1中的PI(4,5)P2结合位点 和 ANO2。初步数据为 PI(4,5)P2 中存在 3 个不同的 PI(4,5)P2 结合位点提供了强有力的支持。 ANO1。 (3) 我们将确定 3 个不同的 PI(4,5)P2 结合位点在调节中的功能作用 ANO1 Ca2+ 敏感性、门控和失活。比较 ANO1 和 ANO2 的一个令人信服的理由是 尽管这 2 个蛋白质在序列上有 70% 相似（57% 相同），但 ANO1 会受到 PI(4,5)P2 的刺激，而 ANO2 被抑制。这种差异提供了丰富的机会来了解 PI(4,5)P2 结合如何与 通道功能。这些研究将回答有关这些监管的紧迫的突出问题 对人类健康和疾病至关重要的渠道。