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）：在健康和疾病中的组织和器官功能取决于宏观中细胞过程的集体活动。器官和组织功能基础的细胞活性取决于对各种化学和电信号范围响应的离子通道组的纳米级分布和合作行为。高分辨率下的离子通道行为仍然鲜为人知，并且在生理环境中以单个通道分辨率研究离子通道集合的分布和合作特性的技术有限。该研究项目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的SICM阵列来实现的，该悬臂具有带有低弹簧常数的尖锐指示尖端，从而可以对软生物膜进行成像。为了获取局部电导传导图像，只有金属尖端顶点才能在悬臂的其余部分完全绝缘进行。每个导电悬臂都将拥有压电致动，允许探针的独立Z翻译。压电量将用作反馈控制，可以控制悬臂运动以保持恒定的电气传导测量。然后将应用这种新的SICM阵列研究吸烟介导的氧化应激对半通道活性的影响。我们的实验室位于使用AFM定义半通道的3D结构和活动的最前沿。半通道在维持离子细胞稳态和传输化学信号方面起着至关重要的作用。可以通过吸烟引起的氧化应激来改变半通道的活性。但是，对于半通道种群的分布和合作特性以及如何通过药物滥用而改变，知之甚少。为了探测氧化应激对半通道的影响，将在定制的纳米多孔硅载体上沉积半通道。支撑系统将安装在两个腔室SICM样品支架上，其中双层的顶部和底部是电分离的，而离子电流只能通过半通道在顶部和底部腔室之间传递。因此，在正常条件下，在多个位置和暴露于粘液脂肪的情况下，例如通过药物滥用产生的氧化剂，尤其是吸烟。我们对药物/药物滥用介导的半通道行为的理解将有助于我们设计预防性和/或治疗方法来滥用药物。在本提案中创建的新型SICM阵列将对电信号和传播进行广泛的应用 并发现跨生理系统的新型治疗剂。