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

描述(申请人提供)：在健康和疾病中，组织和器官的功能是由细胞过程在宏观尺度上的集体活动决定的。作为器官和组织功能基础的细胞活动是由对不同化学和电信号做出反应的离子通道组的纳米级分布和协作行为来定义的。离子通道在高分辨率下的行为仍然知之甚少，在生理环境中研究单通道分辨率的离子通道系综的分布和协作特性的技术也很有限。本研究项目的目的是设计一种新型的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成像。这是通过创建悬臂式SICM阵列来实现的，该阵列具有尖锐的导电尖端和低的弹性常数，从而允许对柔软的生物膜进行成像。为了获得局部导电图像，只有金属尖端将与悬臂的其余部分完全绝缘地导电。每个导电悬臂梁将具有压电驱动，允许探头的独立z平移。压电致动器将作为反馈控制器，控制悬臂梁的运动，以保持恒定的电导测量。然后，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管活性的影响。我们的实验室在使用原子力显微镜定义半管的三维结构和活性方面处于领先地位。半通道在维持细胞内离子平衡和传递化学信号方面起着至关重要的作用。吸烟引起的氧化应激可改变半管的活性。然而，人们对半沟鼠种群的分布和合作特性以及它们如何被药物滥用改变知之甚少。为了探索氧化应激对半隧道的影响，我们将在定制的纳米多孔硅载体上沉积半隧道。支撑系统将安装在两个腔室的SICM样品支架上，其中双层的顶部和底部是电隔离的，离子电流只能通过半通道在顶部和底部的腔体之间流动。因此，导电的SICM阵列将被用来研究在正常条件下和在暴露于诸如药物滥用产生的氧化剂，特别是吸烟产生的氧化剂后，同时在多个位置的半通道的动态行为。我们对药物/物质滥用介导的半通道行为的理解将有助于我们设计预防和/或治疗药物滥用的方法。该方案中创建的新型SICM阵列将在电信号和传播的研究中具有广泛的应用 以及跨生理系统的新疗法的发现。

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.