Molecular Physiology of TMEM16/Anoctamin Proteins

了解小分子跨细胞膜运输的机制是一个 细胞生理学中的根本挑战。这个应用集中在一个转运蛋白家族上，即 AnocTamins/TMEM16s，因为它们在细胞生理学中扮演着不同的和不可或缺的角色。开国大业 Anoctamin(ANO)家族的成员是钙激活的氯离子通道(ANO1和ANO2)。这些渠道 它们无处不在地表达，并密切参与通过推动分泌 体液，控制肠道运动，促进激素分泌，调节神经元兴奋性 以及平滑肌肉的收缩能力，以及其他功能。ANO1功能障碍与多种疾病有关 人类疾病的状态，包括高血压、结肠炎、哮喘和肺部疾病。人类基因的破坏 小鼠的ANO1基因会导致严重的发育异常、行为障碍、胃肠道改变 能动性和感知疼痛的能力。因为ANO1和ANO2在细胞中扮演着多种多样但必不可少的角色 生理学上，它们代表了治疗药物开发的新靶点，但到目前为止还没有药物靶点。 受到的关注相对较少。最近，已经提供了包括ANO1的各种ANO的3-D结构 对这些蛋白质是如何工作的有价值的见解，但主要问题仍然存在。我们研究的长期目标是 了解ANO1(TMEM16A)和ANO2(TMEM16B)的结构和功能。特别是在这一点上 应用：磷脂磷脂酰肌醇-(4，5)二磷酸对ANO1的调节作用 (PI(4，5)P2)。虽然PI(4，5)P2是细胞膜中的一种微量脂类，但显然它起着关键的作用，但作用不大 了解，在ANO1和ANO2功能中的作用。我们将使用单细胞电生理学的组合，定向 突变和计算分子动力学模型来解释如何打开和关闭 ANO1受PI(4，5)P2和钙离子控制。它有三个目标：(1)我们将表征生物物理 PI(4，5)P2调节ANO1和ANO2的机制及PI(4，5)P2与ANO2的功能相互作用 钙，以及磷脂酰肌醇和肌醇磷酸盐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，并在ANO1中定位PI(4，5)P2结合位点 和ANO2。初步数据有力地支持了三种不同的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结合位点在调控中的功能作用 ANO1钙敏感性、门控和失活。比较ANO1和ANO2的一个令人信服的原因是 虽然这两个蛋白在序列上有70%的相似性(57%相同)，但ANO1受到PI(4，5)P2的刺激，而 ANO2被抑制。这种差异为理解PI(4，5)P2结合是如何耦合到 通道功能。这些研究将回答有关监管的紧迫问题。 对人类健康和疾病至关重要的渠道。

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.