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

描述(申请人提供)：本申请的总体目标是设计和开发纳米技术，以研究关键的纳米级生物结构-离子通道和受体，这是所有生命功能所必需的。他们的三维(3D)结构和活动在与生活方式相关的刺激(包括吸毒)的反应下发生变化，会引发严重的健康异常。了解这些纳米生物结构的三维结构-活性关系一直是一个核心但又难以实现的目标。目前，3D结构是用时间和资源限制的X射线衍射和EM来检测的。用膜片钳和荧光显微镜分析离子通道活性。然而，目前还没有一套完整的系统来直接研究这些纳米生物结构在水溶液中的三维构效关系。原子力显微镜(AFM)在水介质中为包括通道和受体在内的许多大分子络合物提供了高分辨率的结构信息。AFM非常适合于成像表面拓扑--包括药物分子在内的外部刺激通常会在其中相互作用的主要结构域。开放的AFM体系结构允许集成其他技术。集成的多模式原子力显微镜将允许实时成像通道(或受体)-刺激(或扰动)复合体、它们的物理化学性质和所产生的通道构象。我们建议设计一种最先进的双腔原子力显微镜，它集成了高分辨率成像和渗透率分析工具。为了测试其与NIDA的使命相关的应用，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)。他们的3D结构和他们对离子和信号分子的渗透性，对药物成瘾诱导刺激的反应将被检查。半通道将细胞与其细胞外环境或其邻近细胞联系起来。它们与吸烟诱导的细胞病理有关，它们的存在受到与药物成瘾相关的细胞受体(如多巴胺受体)和刺激的调节。应用的具体目标是：1.设计一种组合式原子力显微镜，支持带有纳米孔、TIRF、单分子FRET和压敏染料成像系统的硅芯片。作为对该系统的测试，获得了半脑沟和AChR的图像三维结构。2A。检查分子渗透性和离子电导，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)检查确定的门控剂和与药物成瘾相关的扰动的作用，包括吸烟冷凝物、尼古丁和ROS对通道通透性的影响，以及2b。研究药物(如可卡因、尼古丁)和病原体对单细胞质膜半角形核和AChR(一种药物成瘾相关受体)的密度、分布和周转的影响。这项研究开发的集成成像系统将是此类系统中的第一个，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的发展方面具有深远和广泛的作用。 公共卫生相关性：吸毒成瘾对人类健康和整个社会的影响是相当大的，但对这些成瘾的原因和/或有害影响的潜在机制(S)了解有限。大多数药物成瘾刺激可能通过调节离子通道和受体的结构和活性而产生作用，如乙酰胆碱受体(AChR)和缝隙连接半通道。目前还没有实验工具来同时测量离子通道的活性，同时成像其3D分子结构和3D构象；然而，这些信息对于促进我们对药物成瘾的分子机制和/或其病理后果的理解是必不可少的。纳米科学和技术的进步可能为探索由纳米生物结构(如离子通道和受体)介导的复杂病理过程提供了最好的途径。在这里，我们打算实现最先进的集成多模式工具，并在两大类离子通道上测试它们的应用，半通道和AChR。我们成功完成拟议的工作将像是将EM与膜片钳和单分子成像相结合，填补了这一空白，并将为药物成瘾治疗和治疗的开发提供可行的途径。

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.