Gating Mechanisms of Retinal Rod cGMP Activated Channels

描述（申请人提供）：离子通道是精细的分子机器。通过打开和关闭细胞膜上的离子选择孔，这些蛋白质最终控制了从我们的感官到我们的思想的一切。我们的长期目标是了解离子通道门控行为背后的精确分子运动。环核苷酸门控（CNG）通道在我们的光感受器中产生主要的电信号，以响应光。它们是非选择性阳离子通道，通过环核苷酸（cAMP和cGMP）与通道的直接结合而打开，并由各种第二信使调节。除了在视觉中发挥作用外，它们对嗅觉和味觉也很重要，这些通道的突变会导致从失明到嗅觉缺失的各种感觉障碍。它们的动态行为控制着我们的视觉感知，但其功能的分子机制在很大程度上是未知的。这种空白部分是由于缺乏实验方法，使我们能够在原子分辨率下实时“观察”蛋白质的作用。我们的目标是通过开发新的荧光方法来填补这一空白，并将其应用于研究CNG通道的激活和调节机制。我们将利用两个令人兴奋的新发展：1)我们对细胞内配体结合和密切相关的HCN2和SpIH通道的门控域的x射线晶体结构的解决方案，以及2)我们开发的同时从无细胞膜斑块进行电流和荧光测量的方法（称为膜片钳荧光法，PCF）。我们的具体目标是精确确定通道中两个重要部分的分子重排，即环核苷酸结合域和c连接体，该区域将环核苷酸的结合与孔的打开结合。在这些实验结束时，我们将更多地了解CNG和相关通道的工作原理，并将开发出适用于其他通道和其他蛋白质的分子重排研究的新方法。公共卫生相关性：离子通道是大脑的晶体管，因此控制着从我们的感觉到我们的思想的一切。环核苷酸门控（CNG）通道在我们的视杆细胞和视锥细胞中产生主要的电信号来响应光，这些通道的突变会导致从失明到嗅觉丧失等各种感觉障碍。我们的长期目标是了解这些重要蛋白质如何在分子水平上起作用，以揭示蛋白质功能的基本机制，并使我们能够开发针对神经系统疾病的靶向治疗。

DESCRIPTION (provided by applicant): Ion channels are exquisite molecular machines. By opening and closing an ion selective pore across the cell membrane, these proteins ultimately control everything from our senses to our thoughts. Our long term goal is to understand the precise molecular motions that underlie this gating behavior of ion channels. Cyclic nucleotide-gated (CNG) channels produce the primary electrical signal in our photoreceptors in response to light. They are nonselective cation channels that are opened by the direct binding of cyclic nucleotides (cAMP and cGMP) to the channel and modulated by various second messengers. In addition to their role in vision, they are also essential for olfaction and taste, and mutations in these channels cause an assortment of sensory disorders ranging from blindness to anosmia. Their dynamic behavior controls our visual perception, yet the molecular mechanism for their function is largely unknown. This void is due, in part, to a lack of experimental approaches that allow us to "watch" proteins in action in real time at atomic resolution. We aim to fill this void by developing novel fluorescence approaches and applying them to investigate the mechanisms of activation and modulation of CNG channels. We will take advantage of two exciting new developments: 1) our solution of the x-ray crystal structures of the intracellular ligand binding and gating domains of the closely related HCN2 and SpIH channels, and 2) our development of methods for simultaneous current and fluorescence measurements from cell-free membrane patches (termed patch-clamp fluorometry, PCF). Our specific aims are to precisely determine the molecular rearrangement in two important parts of the channel, the cyclic nucleotide- binding domain, and the C-linker, the region that couples binding of cyclic nucleotides to opening of the pore. At the conclusion of these experiments we will know a great deal more about how CNG and related channels work, and will have fully developed new approaches to studying molecular rearrangements applicable to other channels and other proteins. PUBLIC HEALTH RELEVANCE: Ion channels are the transistors of the brain and thereby control everything from our senses to our thoughts. Cyclic nucleotide-gated (CNG) channels produce the primary electrical signal in our rods and cones in response to light, and mutations in these channels cause an assortment of sensory disorders ranging from blindness to anosmia. Our long term goal is to understand how these important proteins work at the molecular level to uncover basic mechanisms of protein function and enable us to develop targeted therapies for neurological diseases.