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。对单细胞分泌的激素和神经递质的量化提供了对这些分子调控的细胞信号过程的动力学和机制的关键见解。碳纤维微电极已被广泛应用于这些研究中，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)以及几种激素(如胰岛素)的快速、高灵敏和无标记测量。这些研究已经获得了与包括抑郁症、成瘾和糖尿病在内的一系列疾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，通常分析的靶标不到10个。发展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，但样品准备最少，这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新的范式。我们建议开发一种新型的生化“嗅探器”传感器，其中重组蛋白嵌合体由连接到G蛋白偶联受体(称为ICCR)的离子通道组成超稳定的平面膜，并由悬浮在玻璃微管上的聚合磷脂双层组成。当ICCR的GPCR亚单位与配体结合和构象激活时，通过ICCR亚单位的离子通量将被调制，并随后通过电生理检测来测量。灵敏度将与配体-受体对的Kd相当，通常在NM-M区域。因此，这项技术将把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)将制备、提纯ICCR，并将其重组为人造脂膜，以优化蛋白质活性的稳定性和保持性。B)将研究人工膜中ICCR的关键结构-活性关系，例如功能性ICCR中IC和GPCR亚单位的取向和化学计量，这些信息将被用于设计更灵敏的ICCR传感器。C)基于含有D2多巴胺和胰高血糖素受体的ICCR的传感器将被制备和评估，分别用于选择性地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标的刺激释放。这项工作的成功完成将为定量检测具有重要生物学意义但在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.