Scanning Ion Conductance Microscope-array for the Study of Ion Channel Clusters

用于研究离子通道簇的扫描离子电导显微镜阵列

• 批准号: 8457361
• 负责人: Brian R Meckes
• 金额: $ 3.49万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-01-14 至 2016-01-13
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8457361
• 关键词: Affect; Architecture; Area; Atomic Force Microscopy; Behavior; Biological; Cell physiology; Cells; Chemicals; Communication; Connexin 43; Connexins; Cooperative Behavior; Coupling; Crystallography; Custom; Deposition; Disease; Drug Addiction; Drug Kinetics; Environment; Exposure to; Extravasation; Feedback; Functional disorder; Funding; Goals; Health; Homeostasis; Human Activities; Image; Imaging Device; Individual; Ion Channel; Ions; Laboratories; Left; Length; Location; Maps; Measurement; Mechanics; Mediating; Membrane; Microscope; Molecular; Molecular Conformation; Motion; Movement; NMR Spectroscopy; Organ; Oxidative Stress; Pathway interactions; Pharmaceutical Preparations; Physiological; Play; Population; Population Dynamics; Preventive; Property; Proteins; Radial; Research; Research Project Grants; Resolution; Role; Sampling; Scanning; Signal Transduction; Silicon; Smoking; Stimulus; Structure; Substance Addiction; Substance abuse problem; Support System; System; Techniques; Technology; Therapeutic; Three-Dimensional Imaging; Tissues; Translations; United States National Institutes of Health; Variant; addiction; biological systems; cantilever; cognitive function; density; design; gap junction channel; imaging modality; macromolecule; nanometer; nanoscale; novel; novel therapeutics; public health relevance; receptor; therapeutic target; three dimensional structure; tool

DESCRIPTION (provided by applicant): Tissue and organ function, in health and disease, is determined by the collective activity of cellular processes at the macroscale. Cellular activity underlying organ and tissue function is defined by the nanoscale distributive and cooperative behavior of groups of ion channels that respond to a diverse range of chemical and electrical signals. Ion channel behavior at high resolution remains poorly understood and there are limited techniques for studying the distributed and cooperative properties of ion channel ensembles with single channel resolution, in a physiological environment. The aim of this research project is to design a novel scanning ion conductance microscope (SICM) probe capable of molecular resolution imaging of the conductance and structure of multiple points simultaneously. This is accomplished through the creation of a cantilevered SICM-array featuring sharp conducting tips with low spring constants, which allow for imaging of soft biological membranes. To get localized electrical conduction images, only the metallic tip apex will be conducting with the remainder of the cantilever completely insulated. Each conducting cantilevers will possess piezoelectric actuation allowing for the independent z translation of the probes. The piezoactuators will serve as feedback controls that can control the cantilever movement to maintain a constant electrical conduction measurement. This new SICM-array will then be applied to study the effect of smoking-mediated oxidative stress on the activity of hemichannels. Our laboratory is at the forefront of defining the 3D structure and activity of hemichannels using AFM. Hemichannels play a critical role in maintaining ionic cellular homeostasis and transmitting chemical signals. The activity of hemichannels can be altered through oxidative stress caused by smoking. However, little is known about the distributive and cooperative properties of populations of hemichannels and how they are altered by substance abuse. To probe the effect of oxidative stress on hemichannels, hemichannels will be deposited on custom fabricated nanoporous silicon supports. The support system will be mounted on a two chamber SICM sample holder, in which the top and bottom of the bilayers are electrically separated and ionic current can only pass between the top and bottom chamber through hemichannels. Thus, the conducting SICM-array will be utilized to study the dynamic behavior of hemichannels in multiple locations simultaneously in normal conditions and following exposure to peturbants, such as oxidative agents produced by substance abuse, particularly smoking. Our understanding of drug/substance abuse-mediated hemichannel behavior will help us design preventive and/or therapeutic approaches for substance abuse. The novel SICM-array created in this proposal will have broad applications for the study of electrical signaling and propagation and discovery of novel therapeutics across physiological systems.

描述（由申请人提供）：健康和疾病中的组织和器官功能是由宏观尺度上细胞过程的集体活动决定的。器官和组织功能的细胞活动是由纳米级离子通道群的分布和合作行为来定义的，这些离子通道对各种化学和电信号做出反应。高分辨率下的离子通道行为仍然知之甚少，并且在生理环境中研究单通道分辨率离子通道群的分布和协同特性的技术有限。本课题的目的是设计一种新型扫描离子电导显微镜（SICM）探针，能够同时对多个点的电导和结构进行分子分辨率成像。这是通过创建一个悬臂式sicm阵列来实现的，该阵列具有具有低弹簧常数的尖锐导电尖端，可以对软生物膜进行成像。为了获得局部导电图像，只有金属尖端尖端将导电，其余的悬臂完全绝缘。每个导电悬臂梁将具有压电驱动，允许探针的独立z平移。压电致动器将作为反馈控制器，可以控制悬臂运动，以保持恒定的电导测量。这种新的sicm阵列将被应用于研究吸烟介导的氧化应激对半通道活性的影响。我们的实验室在使用AFM定义半通道的3D结构和活性方面处于领先地位。半通道在维持离子细胞稳态和传递化学信号方面起着关键作用。半通道的活性可以通过吸烟引起的氧化应激而改变。然而，人们对半通道种群的分布和合作特性以及它们如何被药物滥用所改变知之甚少。为了研究氧化应激对半通道的影响，将半通道沉积在定制的纳米多孔硅载体上。支撑系统将安装在一个双腔SICM样品支架上，其中双层的顶部和底部是电分离的，离子电流只能通过半通道在顶部和底部之间通过。因此，导电sicm阵列将被用于研究在正常条件下同时在多个位置的半通道的动态行为，以及暴露于刺激物（如药物滥用，特别是吸烟产生的氧化剂）后的动态行为。我们对药物/物质滥用介导的半通道行为的理解将帮助我们设计药物滥用的预防和/或治疗方法。本文提出的新型sicm阵列将在电信号和传播的研究中具有广泛的应用前景