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

描述（由申请人提供）：健康和疾病中的组织和器官功能由宏观尺度上细胞过程的集体活动决定。作为器官和组织功能基础的细胞活性由离子通道组的纳米级分布和协作行为定义，所述离子通道组响应于不同范围的化学和电信号。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，用于研究具有单通道分辨率的离子通道集合的分布和合作性质的技术有限。本研究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率成像。这是通过创建悬臂SICM阵列来实现的，该悬臂SICM阵列具有具有低弹簧常数的尖锐导电尖端，其允许对软生物膜进行成像。为了获得局部导电图像，只有金属尖端顶点将导电，而悬臂的其余部分完全绝缘。每个导电杠杆将具有压电致动，允许探针的独立z平移。压电致动器将作为反馈控制，可以控制悬臂的运动，以保持恒定的导电测量。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结构和活动方面处于领先地位。半通道在维持细胞离子稳态和传递化学信号中起着关键作用。半通道的活性可以通过吸烟引起的氧化应激而改变。然而，很少有人知道的分配和合作性质的人口的hemichannels，以及它们是如何改变药物滥用。为了探测氧化应激对半通道的影响，将半通道沉积在定制制造的纳米多孔硅载体上。支持系统将安装在双室SICM样品保持器上，其中双层的顶部和底部是电分离的，并且离子电流只能通过半通道在顶部和底部室之间通过。因此，导电SICM阵列将被用来研究在正常条件下和暴露于扰动剂（例如由物质滥用，特别是吸烟产生的氧化剂）之后同时在多个位置中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药物滥用的预防和/或治疗方法。该方案中产生的新型SICM阵列将在电信号和传播的研究中具有广泛的应用 以及发现跨生理系统的新疗法。

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.