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

基于离子通道-GPCR 嵌合体和多脂膜的嗅探生物传感器

• 批准号: 8661576
• 负责人: CRAIG A ASPINWALL
• 金额: $ 37.56万
• 依托单位: UNIVERSITY OF ARIZONA
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-09-30 至 2016-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8661576
• 关键词: Address; Agonist; Area; Artificial Membranes; Binding; Binding Sites; Biochemical; Biological Assay; Biosensor; Cataloging; Catalogs; Cell Signaling Process; Cell secretion; Cells; Chimera organism; Chimeric Proteins; Coupled; Detection; Development; Diabetes Mellitus; Disease; Dopamine; Dopamine D2 Receptor; Elements; Environment; Evaluation; Exocytosis; Fluorescence Microscopy; Funding; G-Protein-Coupled Receptors; Glass; Glucagon; Glucagon Receptor; Hormones; Human; In Vitro; Insulin; Investigation; Ion Channel; Ion Channel Protein; Ions; Islets of Langerhans; Knowledge; Label; Ligand Binding; Ligands; Link; Lipids; Liquid substance; Measurement; Measures; Membrane; Membrane Lipids; Mental Depression; Methods; Microelectrodes; Molecular; Monitor; Neuroendocrine Cell; Neuromodulator; Neurotransmitters; Norepinephrine; Opioid; Opioid Receptor; Optics; Performance; Phospholipids; Positioning Attribute; Preparation; Prevention; Property; Proteins; Recombinant Proteins; Regulation; Research Project Grants; Resolution; Role; Rupture; Sampling; Serotonin; Signal Pathway; Signal Transduction; Signaling Molecule; Structure-Activity Relationship; System; Technology; Transducers; Work; Xenopus oocyte; Yeasts; addiction; base; carbon fiber; design; in vivo; insight; instrument; monomer; novel; paracrine; polymerization; public health relevance; receptor; receptor binding; receptor coupling; receptor function; receptor structure function; reconstitution; sensor; stoichiometry; tool

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.

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有关，包括糖尿病、抑郁症和许多其他疾病。单细胞分泌的激素和神经递质的定量为了解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重要的见解。碳纤维微电极已广泛应用于这些研究中，能够快速、高灵敏度、无标记地测量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（例如胰岛素）。这些研究得出了与抑郁症、成瘾和糖尿病等一系列疾病相关的信号传导机制的关键信息。不幸的是，可通过伏安法和安培法测定的细胞分泌产物的数量非常有限，通常分析的目标不到 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，将能够研究更大范围的激素和神经调节剂，并将在细胞信号传导研究中建立新的范例。 我们建议开发一类新型生化“嗅探器”传感器，其中重组蛋白嵌合体由与 G 蛋白偶联受体（称为 ICCR）缀合的离子通道组成，被重构为超稳定的平面膜，并由悬浮在玻璃微管上的聚合磷脂双层组成。 ICCR 的 GPCR 亚基与配体结合和构象激活后，通过 IC 亚基的离子通量将受到调节，随后通过电生理检测进行测量。灵敏度与配体-受体对的 Kd 相当，通常在 nMM-M 范围内。因此，这项技术将把无标记的电生理学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、纯化并重构为人工脂质膜，该膜针对稳定性和蛋白质活性的保留进行了优化。 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 亚基的方向和化学计量，并且该信息将用于设计更具响应性的 ICCR 传感器。 c) 将制备和评估基于包含 D2 多巴胺和胰高血糖素受体的 ICCR 的传感器，用于分别选择性测量多巴胺和胰高血糖素分泌，然后用于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为定量检测具有生物学意义但分析上具有挑战性的细胞释放物提供强大的新工具。