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广泛的人类疾病有关，包括糖尿病、抑郁症和许多其他疾病。单细胞中激素和神经递质分泌的定量为这些分子调节的细胞信号传导过程的动力学和机制提供了关键的见解。碳纤维微电极已广泛应用于这些研究中，以实现几种电活性神经递质（多巴胺、血清素、去甲肾上腺素等）以及一些激素（如胰岛素）的快速、高灵敏度和无标记测量。这些研究已经获得了与一系列疾病（包括抑郁症、成瘾和糖尿病）相关的信号机制的关键信息。不幸的是，可以通过伏安法和安培法检测的细胞分泌产物的数量非常有限，通常分析的靶点不到10个。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，但样品制备最少，将使研究更大范围的激素和神经调节剂成为可能，并将在细胞信号研究中建立一个新的范例。我们建议开发一种新型的生化“嗅探”传感器，其中重组蛋白嵌合体由离子通道偶联到g蛋白偶联受体（称为ICCR）组成，重构成超稳定的平面膜，并由悬浮在玻璃微移液管上的聚合磷脂双层组成。在ICCR的GPCR亚基的配体结合和构象激活后，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体制下。因此，这项技术将扩展无标记，电生理检测到缺乏光学或电化学活性的细胞信号分子。在项目期间：a)制备、纯化ICCRs，并将其重组为人工脂质膜，以优化其稳定性和保持蛋白质活性。b)研究人工膜中ICCR的关键结构-活性关系，如功能性ICCR中IC和GPCR亚基的取向和化学计量，这些信息将用于设计更灵敏的ICCR换能器。c)基于含有D2多巴胺和胰高血糖素受体的ICCRs的传感器将被制备和评估，分别用于选择性测量多巴胺和胰高血糖素的分泌，然后用于监测单细胞对这些靶点的刺激释放。这项工作的成功完成将为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提供一个强大的新工具。

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.