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，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）结构和活动的变化，响应与生活方式有关的刺激，包括药物成瘾，引发严重的健康异常。了解这些纳米生物结构的3D结构-活性关系一直是一个核心但又难以捉摸的目标。目前，3D结构的检查与时间和资源限制的X射线衍射和EM。通过膜片钳和荧光显微镜分析离子通道活性。然而，目前还没有一个完整的系统，直接三维结构的活性研究，这些纳米生物结构在水性缓冲液。原子力显微镜（AFM）提供了高分辨率的结构信息，在水介质中，许多大分子复合物，包括通道和受体。AFM非常适合对表面拓扑结构进行成像-外部刺激（包括药物分子）通常会相互作用的主要结构域。AFM的开放式体系结构允许其他技术的集成。一个集成的多模态AFM将允许实时成像的通道（或受体）刺激（或扰动）复杂，其物理化学性质和产生的通道构象。我们建议设计一个国家的最先进的双室原子力显微镜集成高分辨率成像和渗透性测定工具。作为一个测试其应用程序相关的NIDA的使命，这一应用程序的潜在支持者，我们将研究两个重要的离子通道：半通道和乙酰胆碱受体（AChR），是密切相关的药物成瘾。他们的3D结构和他们的渗透性离子和信号分子，在响应药物成瘾诱导刺激将被检查。半通道将细胞连接到其细胞外环境或其相邻细胞。它们与吸烟诱导的细胞病理学有关，并且它们的存在受到药物成瘾相关细胞受体（例如，多巴胺受体）和刺激物。申请的具体目的是：1。设计了一套组合式AFM、支撑纳米孔硅芯片、TIRF、单分子FRET和电压敏感染料成像系统。作为对该系统的测试，对半通道和AChR的三维结构进行了成像。2a.检查分子渗透性和离子电导，对生理和药物成瘾相关刺激的反应。这包括，a）测量离子、传感器染料和信号分子的通道渗透性，和B）检查限定的门控剂和药物成瘾相关扰动（包括吸烟冷凝物、尼古丁和ROS）对通道渗透性的作用，和2 B。检查单细胞质膜中半通道和AChR（一种药物成瘾相关受体）响应药物（例如，可卡因、尼古丁）和病理性物质。在这项研究中开发的集成成像系统将是第一个同类产品，并将有深远的和更广泛的作用，在定义我们的药物成瘾的分子决定因素的理解，他们的病理后果，以及在药物成瘾和治疗的治疗方法的发展。 公共卫生相关性：药物成瘾对人类健康和整个社会的后果是相当大的，但对这些成瘾的原因和/或有害影响的潜在机制的理解有限。大多数药物成瘾刺激物可能通过调节离子通道和受体如乙酰胆碱受体（AChR）和缝隙连接半通道的结构和活性而发挥作用。目前还没有实验工具来同时测量离子通道活性，同时成像其3D分子结构，3D构象;然而，这是一种信息，对于促进我们对药物成瘾和/或其病理后果的分子机制的理解至关重要。纳米科学和技术的进步可能为探索由纳米级生物结构（如离子通道和受体）介导的复杂病理过程提供了最佳途径。在这里，我们打算实现最先进的集成多模式工具，并测试其应用程序的两个主要类别的离子通道，半通道和乙酰胆碱受体。我们成功完成拟议的任务将像EM与膜片钳和单分子成像相结合，填补了空白，并将为药物成瘾和治疗的治疗方法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.