Structure, Activity & Physiological Role of Hemichannels

描述（由申请人提供）：离子通道和受体是介导关键生理和病理活性的膜界面大分子。了解它们的三维结构-活性关系一直是生物药理学家的核心目标，但却难以实现。纳米尺寸和结构的复杂性，如离子通道的亚基结构和中央渗透孔，它们的膜插入和脂质-蛋白质相互作用对其结构分析提出了主要限制。离子通道的高分辨率3D结构主要用X射线衍射和电子显微镜检查。离子通道活性通常通过膜片钳和荧光显微镜来分析。然而，将这些技术整合到用于离子通道的直接结构-功能活性的组合系统中是有限的。原子力显微镜（AFM）提供了许多生物大分子，包括离子通道和受体的高分辨率结构信息。原子力显微镜对表面、配体和其他门控剂相互作用并改变通道活性的主要结构域进行成像。AFM允许在线添加药理学或（病理）生理刺激。其开放式架构允许与其他技术集成，允许通道刺激复合物成像，从而产生3D构象和通道特性，如渗透性，电导，能量学和力学。本申请的主要焦点是两方面的：a）具有带有支持芯片的实用集成双室AFM，将它们与高灵敏度电学和荧光测量工具联合收割机结合，以及B）使用它们来研究关于两个不同通道的结构-活性关系的假设驱动的机制问题：i）用于高分辨率和灵活性工作的连接蛋白半通道，特别是其细胞质面，对于其知之甚少但推测很多，以及对于分子渗透性和ii）形成良好的高电导pH门控通道的KcsA通道。半通道将用于结构和渗透性研究。KcsA通道将用于测试电导、通道表面形貌以及通道打开和关闭中涉及的束交叉的打开的同时测量。本申请的具体目标是：目标1：设计具有单个或多个纳米孔的支持芯片，并将其与高分辨率AFM集成。目的2：重建半通道和全长或截短的KcsA通道的3D形貌成像。目的3：研究通道亚基结构变化在开放/关闭功能状态中的作用。这包括结构的高分辨率成像，同时测量半通道特异性染料和大分子的渗透性，集成TIRF显微镜和检查各种已知的门控剂，定点肽和抗体对渗透性的作用。对于KcsA通道，将研究截断通道和全长通道-用于3D结构成像的截断通道和用于成像束交叉以关联通道门控的全长通道。目标1和目标3在一定程度上是相互依赖的，而目标2是独立的，仅依赖于设计具有低弹簧常数和高S/N比的尖锐AFM针尖。公共卫生相关性：离子通道和受体是决定关键生理功能的膜结构。其结构和活性的异常是人类主要疾病的基础，但对其结构-活性关系的理解有限。目前还没有实验工具可以在对离子通道的三维结构进行成像的同时测量离子通道的活性，但这是促进我们对人类疾病分子机制的理解所必需的信息。我们的研究的长期意义是相当大的。在我们的研究中开发的技术，即结合等效的EM和单通道电导（膜片钳）和渗透能力将提供一个重大突破的三维结构功能研究的离子通道和受体。我们对半通道和钾通道的研究结果将为研究细胞-细胞外交换机制及其在调节组织稳态中的作用提供新的范式。从我们的研究中获得的机制信息将用于设计有效的预防和治疗疾病，包括心律失常，神经退行性疾病和癌症。

DESCRIPTION (provided by applicant): Ion channels and receptors are membrane interface macromolecules that mediate key physiological and pathological activity. Understanding their three-dimensional structure-activity relationship has been a central and yet elusive goal of biophysicists. The nanoscale size and structural complexity, such as an ion channel's subunit architecture and the central permeable pore, their membrane insertion and lipid-protein interactions put major constraints on their structural analysis. High resolution 3D structure of ion channels is being examined primarily with X-ray diffraction and electron microscopy. Ion channel activity is commonly analyzed by patch clamping and fluorescence microscopy. However, integration of these techniques into a combined system for the direct structure-function activity of ion channels is limited. Atomic force microscopy (AFM) provides high resolution structural information for many biological macromolecules, including ion channels and receptors. AFM images surfaces, the primary structural domains where ligands and other gating agents interact and alter channel activity. AFM allows online addition of pharmacologic or (patho-) physiologic stimuli. Its open architecture allows integration with other techniques permitting imaging of channel-stimuli complex, resulting 3D conformations and channel properties such as permeability, conductance, energetics, and mechanics. The main focus of this application is two-fold: a) to have a practical integrated double chamber AFM with the Support Chip, combine them with high sensitive electrical and fluorescence measuring tools and b) to use them to study hypothesis-driven mechanistic questions about the structure-activity relation for two different channels: i) Connexin hemichannels for high resolution and flexibility work, especially its cytoplasmic face, for which very little is known but much is speculated and for molecular permeability and ii) KcsA channels that form nice, high-conductance pH gated channels. Hemichannel will be used for structural and permeability study. KcsA channel will be used to test simultaneous measurement of conductance, the channel surface topography, and the opening of the bundle crossing implicated in channel opening and closing. Specific Aims of the application are: Aim 1: Design Support Chip with single or multiple nanopore(s) and integrate it with a high resolution AFM. Aim 2: Image 3D topography of reconstituted hemichannels and full-length or truncated KcsA channels. Aim 3: Examine the role of channel subunit structural changes in the open/closed functional states. This includes high resolution imaging of the structure while simultaneously measuring permeability, of dyes and large molecules specific to hemichannels, with integrated TIRF microscopy and examining role of various known gating agents, site-directed peptides and antibodies on the permeability. For KcsA channels, both truncated as well as full- length channels will be studied - truncated channel for 3D structure imaging and the full-length channel for imaging bundle crossing to correlate channel gating. While Aims 1 and 3 are interdependent to some extent, Aim 2 is independent and only relies on designing sharp AFM tips with low spring constant and high S/N ratio. PUBLIC HEALTH RELEVANCE: Ion channels and receptors are membrane structures that determine key physiological functions. Abnormality in their structure and activity underlie major human diseases, yet there is a limited understanding of their structure-activity relation. Currently there is no experimental tool to measure simultaneously an ion channel activity while imaging its 3D structure, yet this is the kind of information that is essential to advance our understanding of the molecular mechanism underlying human diseases. The long term significance of our study is considerable. The techniques developed in our study, namely combining an equivalent of EM and single channel conductance (patch clamping) and permeability capability will provide a major breakthrough for 3D structure-function study of ion channels and receptors. Our results from study of hemichannels and potassium channel will provide new paradigms to examine the mechanism of cell-extracellular exchanges and their role in modulating tissue homeostasis. Mechanistic information gained from our study will then be used for designing effective prevention and treatment of diseases, including cardiac arrhythmias, neurodegenerative diseases, and cancer.