Sniffer Biosensors Based on Ion Channel-GPCR Chimeras and Polylipid Membranes

描述（由申请人提供）：体内激素和神经递质水平的调节缺陷与多种人类疾病相关，包括糖尿病、抑郁症和许多其他疾病。单细胞激素和神经递质分泌的定量分析为了解这些分子调控的细胞信号传导过程的动力学和机制提供了关键的见解。碳纤维微电极已被广泛用于这些研究中，以实现对几种电活性神经递质（多巴胺、5-羟色胺、去甲肾上腺素等）的快速、高灵敏度和无标记测量，以及一些激素（例如胰岛素）。这些研究已经产生了关于与一系列疾病相关的信号机制的关键信息，包括抑郁症，成瘾和糖尿病。不幸的是，可以通过伏安法和安培法测定的细胞分泌产物的数量是非常有限的，通常分析不到10个目标。开发一种以无标记方式快速检测更广泛的激素和神经递质的能力，具有高灵敏度，选择性和时间分辨率，但样品制备量最少，将能够调查更大的激素和神经调质目录，并将在细胞信号研究中建立一个新的范式。 我们建议开发一类新型的生化“嗅探器”传感器，其中重组蛋白嵌合体，包括一个离子通道共轭的G-蛋白偶联受体（简称为ICCR），重组成一个超稳定的平面膜，并由一个聚合的磷脂双层，悬浮在玻璃微量移液管。在配体结合和ICCR的GPCR亚基的构象活化后，通过IC亚基的离子通量将被调节，随后通过电生理检测进行测量。灵敏度将与配体-受体对的Kd相当，通常在nM-M方案中。因此，该技术将无标记的电生理检测扩展到缺乏光学或电化学活性的细胞信号分子。在项目期间：a）ICCR将被制备、纯化并重构到人工脂质膜中，所述人工脂质膜针对蛋白质活性的稳定性和保留进行了优化。B）将检查人工膜中ICCR的关键结构-活性关系，例如功能性ICCR中IC和GPCR亚基的取向和化学计量，并且该信息将用于设计更响应的ICCR换能器。c）将制备基于含有D2多巴胺和胰高血糖素受体的ICCR的传感器，并评估其分别用于选择性地测量多巴胺和胰高血糖素分泌，然后实施以监测这些靶标从单细胞的刺激释放。这项工作的成功完成将提供一个强大的新工具，用于定量检测生物学上重要但分析上具有挑战性的细胞释放物。 公共卫生相关性：公共卫生相关性：该研究项目将通过开发用于检测单细胞激素和神经递质分泌的新型、最先进的生物传感器，在生化分析中创造一种新的范式。这些化合物的调节缺陷与许多广泛的、使人衰弱的疾病（包括抑郁症和糖尿病）的发展和进展直接相关。该项目开发的生物传感器将能够以无标记的方式快速，灵敏和选择性地检测激素和神经递质，从而更有效地研究这些分子在细胞信号传导途径中的作用，并最终更有效地预防和/或治疗相关疾病。

DESCRIPTION (provided by applicant): Defective regulation of in vivo hormone and neurotransmitter levels is associated with a wide range of human disorders, including diabetes, depression, and numerous others. Quantification of hormone and neurotransmitter secretion from single cells has provided key insights into the dynamics and mechanisms of cell signaling processes regulated by these molecules. Carbon fiber microelectrodes have been widely used in these studies to enable rapid, highly sensitive, and label-free measurement of several electroactive neurotransmitters (dopamine, serotonin, norepinephrine, etc.), as well as a few hormones (e.g. insulin). These studies have yielded key information regarding the signaling mechanisms associated with a range of disorders including depression, addiction, and diabetes. Unfortunately, the number of cellular secretory products that can be assayed via voltammetric and amperometric methods is highly limited, with less than 10 targets commonly analyzed. Developing a capability to rapidly detect a wider range of hormones and neurotransmitters in a label-free manner, with high sensitivity, selectivity and temporal resolution but minimal sample preparation, would enable investigation of a larger catalog of hormones and neuromodulators, and would establish a new paradigm in studies of cell signaling. We propose to develop a novel class of biochemical "sniffer" sensors in which a recombinant protein chimera, comprised of an ion channel conjugated to a G-protein coupled receptor (referred to as an ICCR), is reconstituted into an ultrastable planar membrane, and composed of a polymerized phospholipid bilayer that is suspended across a glass micropipet. Upon ligand binding and conformational activation of the GPCR subunit of the ICCR, ion flux through the IC subunit will be modulated and subsequently measured via electrophysiological detection. The sensitivity will be comparable to the Kd for the ligand-receptor pair, typically in the nM- M regime. Thus this technology will extend label-free, electrophysiological detection to cell signaling molecules that lack optical or electrochemical activity. During the project period: a) ICCRs will be prepared, purified, and reconstituted into artificial lipid membranes that are optimized for stability and retention of protein activity. b) Key structure-activity relationships of ICCRs in artificial membranes will be examined, such as the orientation and stoichiometry of the IC and GPCR subunits in functional ICCRs, and this information will be used to design more responsive ICCR transducers. c) Sensors based on ICCRs containing the D2 dopamine and glucagon receptors will be prepared and evaluated for selectively measuring dopamine and glucagon secretion, respectively, and then implemented to monitor stimulated release of these targets from single cells. Successful completion of this work will provide a powerful new tool for quantitative detection of biologically important yet analytically challenging cellular releasates. PUBLIC HEALTH RELEVANCE: Public Health Relevance: This research project will create a new paradigm in biochemical analysis by developing novel, state-of-the-art biosensors for detecting hormone and neurotransmitter secretion from single cells. Defective regulation of these compounds is directly related to development and progression of a number of widespread, debilitating diseases, including depression and diabetes mellitus. The biosensors developed in this project will enable rapid, sensitive and selective detection of hormones and neurotransmitters in a label-free manner, leading to more effective methods of studying the roles of these molecules in cell signaling pathways, and ultimately to more effective prevention and/or treatment of related diseases.