Designing an Integrated Nanoscale System for Ion Channel Structure-Function Study

描述（由申请人提供）：本申请的总体目标是设计和开发纳米技术来研究关键的纳米级生物结构-离子通道和受体，它们是所有生命功能所必需的。它们的三维（3D）结构和活动的变化是对与生活方式有关的刺激的反应，包括吸毒成瘾，引发严重的健康异常。了解这些纳米生物结构的三维构效关系一直是一个中心但难以实现的目标。3D结构目前用时间和资源限制的x射线衍射和EM进行检查。离子通道活性通过贴片夹紧和荧光显微镜进行分析。然而，目前还没有一个完整的系统来直接研究这些纳米生物结构在水缓冲液中的三维结构-活性。原子力显微镜（AFM）提供了水介质中许多大分子复合物（包括通道和受体）的高分辨率结构信息。AFM非常适合于表面拓扑成像——包括药物分子在内的外部刺激通常会相互作用的主要结构域。AFM的开放架构允许其他技术的集成。集成的多模态AFM将允许通道（或受体）-刺激（或扰动）复合物的实时成像，它们的物理化学性质和产生的通道构象。我们建议设计一个最先进的双室原子力显微镜，集成高分辨率成像和渗透率分析工具。作为与NIDA任务相关的应用测试，我们将研究与药物成瘾密切相关的两个重要离子通道：半通道和乙酰胆碱受体（AChR）。它们的三维结构和它们对离子和信号分子的渗透性，在药物成瘾诱导刺激下的反应将被检查。半通道将细胞与其细胞外环境或邻近细胞连接起来。它们与吸烟引起的细胞病理有关，它们的存在受药物成瘾相关的细胞受体（如多巴胺受体）和刺激的调节。该应用程序的具体目的是：1。设计一个组合AFM，支持硅芯片与纳米孔，TIRF，单分子FRET和电压敏感染料成像系统。作为该系统的测试，对半通道和AChR的三维结构进行了成像。2 a。检查分子渗透率和离子电导，以响应生理和药物成瘾相关的刺激。这包括：a)测量通道对离子、传感器染料和信号分子的通透性；b)检查定义的门控剂和药物成瘾相关的扰动（包括吸烟冷凝物、尼古丁和活性氧）对通道通透性的作用；2b。检查单细胞质膜中半通道和AChR（一种药物成瘾相关受体）对药物（如可卡因、尼古丁）和病理因子的反应的密度、分布和周转。在这项研究中开发的综合成像系统将是同类中的第一个，并将在定义我们对药物成瘾的分子决定因素，其病理后果以及药物成瘾治疗和治疗方法的发展方面发挥深远而广泛的作用。

DESCRIPTION (provided by applicant): Overall goal of this application is to design and develop nanotechnology to study key nanoscale biostructures - Ion channels and receptors that are essential for all living functions. Changes in their three-dimensional (3D) structure and activity, in response to stimuli related to life style, including drug addiction, trigger severe health abnormalities. Understanding 3D structure-activity relationship of these nano-biostructures has been a central and yet elusive goal. 3D structure is currently examined with time and resource limiting X-ray diffraction and EM. Ion channel activity is analyzed by patch clamping and fluorescence microscopy. However, there is no integrated system for a direct 3D structure-activity study of these nano-biostructures in aqueous buffer. Atomic force microscopy (AFM) provides high resolution structural information, in aqueous medium, for many macromolecular complexes, including channels and receptors. AFM is ideally suited to image the surface topology - the primary structural domain where external stimuli, including drug molecules would normally interact. Open architecture of AFM permits integration of other techniques. An integrated multimodal AFM would allow real-time imaging of channel (or receptor)-stimuli (or perturbants) complex, their physicochemical properties and resulting channel conformations. We propose to design a state-of-the-art double chamber AFM integrated with high resolution imaging and permeability assay tools. As a test of its applications related to NIDA's mission, a potential supporter of this application, we will study two important ion channels: hemichannels and Acetyl choline receptor (AChR) that are intimately related to drug addiction. Their 3D structure and their permeability to ions and signaling molecules, in response to drug addiction-inducing stimuli will be examined. Hemichannels connect a cell to its extracellular milieu or its neighbor cells. They are linked to smoking-induced cell pathology and their presence is modulated by drug addiction-related cell receptors (e.g., dopamine receptor) and stimuli. Specific Aims of the application are: 1. Design a combined AFM, Support silicon Chip with a nanopore, TIRF, Single molecule FRET and voltage-sensitive dye imaging systems. As a test of this system, image 3D structure of hemichannels and AChR. 2a. Examine molecular permeability and ionic conductance, in response to physiological and drug addiction-related stimuli. This includes, a) measuring channel permeability to ions, sensor dyes and signaling molecules and b) examining role of defined gating agents and drug addiction-related perturbations, including smoking condensate, nicotine and ROS on the channel permeability, and 2b. Examine density, distribution and turnover of hemichannels and AChR (a drug addiction related receptor) in single cell plasma membrane in response to drugs (e.g., cocaine, nicotine) and pathological agents. The integrated imaging system developed in this study will be first of its kind and will have far reaching and broader role in defining our understanding of the molecular determinants of drug addiction, their pathological consequences as well as in development of therapeutics for drug addiction and treatment. PUBLIC HEALTH RELEVANCE: Consequences of drug addictions on human health and society at large are considerable, yet there is a limited understanding of the underlying mechanism(s) of the cause and/or deleterious effects of these addictions. Most of the drug addiction stimuli possibly induce their effects through them modulating the structure and activity of ion channels and receptors such as acetyl choline receptor (AChR) and gap junctional hemichannels. Currently there is no experimental tool to measure simultaneously an ion channel activity while imaging its 3D molecular structure, the 3D conformations; yet this is the kind of information that is essential to advance our understanding of the molecular mechanism underlying drug addiction and/or their pathological consequences. Advances in nanoscience and technology provide perhaps, the best avenue to explore complex pathological processes that are mediated by nanoscale biostructures, such as ion channels and receptors. Here we intend to implement the most advanced integrated multimodal tools and test their applications on two major classes of ion channels, hemichannels and AChR. Our successful completion of the proposed undertaking will be like combining EM with patch clamping and single molecule imaging that fill the void as well as will provide viable avenues for development of therapeutics for drug addiction and treatment.