A Genetically-Targeted Molecular Wire Fluorescent Sensor for Monitoring Voltage

用于监测电压的基因靶向分子线荧光传感器

• 批准号: 8117708
• 负责人: Evan Walker Miller
• 金额: $ 2.12万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-08-01 至 2012-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8117708
• 关键词: Action Potentials; Aniline; Behavior; Binding; Cells; Cellular Membrane; Characteristics; Chemicals; Chimeric Proteins; Communication; Dependence; Detection; Development; Dyes; Electron Transport; Electrons; Electrophysiology (science); Enzymes; Event; Fluorescence; Fluorescent Probes; Genetic; Hippocampus (Brain); Humulus; Investigation; Kinetics; Label; Length; Life; Long-Term Potentiation; Measurement; Membrane; Methods; Molecular; Molecular Target; Monitor; Neurodegenerative Disorders; Neurologic; Neurons; Noise; Optical Methods; Optics; Population; Process; Rattus; Reaction Time; Research; Resolution; Sampling; Signal Transduction; Solutions; System; Techniques; Xanthenes; attenuation; base; brain cell; combat; electric field; electrical potential; fluorophore; improved; interest; optical imaging; public health relevance; response; sensor; uptake; voltage

DESCRIPTION (provided by applicant): Monitoring electrical activity in neurons is of critical importance for understanding the dynamic manner in which brain cells communicate. Currently, electrophysiology can provide exquisitely precise measurements of voltage changes and action potentials in a single cell, but the spatial information derived from electrophysiological measurement is limited. Optical imaging provides an attractive solution for monitoring voltage changes in multiple neurons simultaneously. Unfortunately, no single optical probe for voltage has been able to supply the requisite characteristics of a probe suitable for sensitive detection of action potentials and other sub-threshold events. In this research, a method is devised for detecting changes in voltage in neuronal cells which incorporates large fluorescence changes (1-2%/mV), fast kinetics (submicrosecond), negligible capacitative load on the cell, genetic targetability, and synthetic tractability. The efficiency of photo- induced electron transfer (PeT) between an aniline donor and a xanthene-based fluorophore acceptor will be altered in the presence of an applied electric field. The magnitude of the change will be proportional to the strength of the field and the length of the dipole formed upon excitation to the singlet excited state. Given that the electric field in a neuronal context will be relatively constant at approximately 105 V/cm, large changes in fluorescence upon application of an electric field can be realized by increasing the dipole, or the distance from donor to acceptor. To combat the exponential distance dependence of electron transfer, which will significantly impair the ability of a PeT-based sensor to respond to voltage, a molecular wire will be used to connect donor to acceptor. Molecular wires decrease the distance dependence of electron transfer, by changing the mechanism of electron transfer from superexchange at close distances to electron hopping at very large distances. A molecular wire will allow effective electron transfer while maximizing the change in fluorescence upon introduction of an electric field. The sensor can be further improved by targeting the probe, through the use of a chemically reactive handle, to genetically defined neuronal sub-populations. Expression of membrane- bound fusion proteins that covalently self-ligate orthogonal chemical handles to themselves will allow for targeting of the sensor to specific sub-types of neurons, increasing the signal-to-noise ratios of the probe in response to voltage changes and allowing for investigation of genetic sub-types of neurons within heterogeneous samples. Development of a fluorescent probe for voltage which can deliver large fluorescence increases in response to changes in voltage while maintaining good temporal fidelity and avoiding capacitative loading will be of broad interest for studying a wide range of neurological systems. PUBLIC HEALTH RELEVANCE: Optical imaging of changes in electrical potential in neuronal cells offers an attractive method for studying the dynamics of neuronal communication. Traditional optical methods for monitoring neuronal activity suffer from low signal-to-noise ratios in response to changes in voltage across the cellular membrane. This research will develop new molecular wire-based fluorescent probes for optically monitoring voltage in living cells with high spatial and temporal resolution.

描述（由申请人提供）：监测神经元中的电活动对于理解脑细胞通信的动态方式至关重要。目前，电生理学可以提供单个细胞中电压变化和动作电位的精确测量，但从电生理测量得到的空间信息是有限的。光学成像为同时监测多个神经元中的电压变化提供了有吸引力的解决方案。不幸的是，没有一个用于电压的光学探针能够提供适合于动作电位和其他亚阈值事件的灵敏检测的探针的必要特性。在这项研究中，设计了一种用于检测神经元细胞中电压变化的方法，该方法结合了大的荧光变化（1-2%/mV）、快速动力学（亚微秒）、细胞上可忽略的电容负荷、遗传靶向性和合成易处理性。苯胺供体和基于咕吨的荧光团受体之间的光诱导电子转移（PeT）的效率将在施加的电场存在下改变。变化的幅度将与场的强度和在激发到单重激发态时形成的偶极子的长度成比例。假设在神经元环境中的电场将相对恒定在约105 V/cm，则在施加电场时荧光的大的变化可以通过增加偶极子或从供体到受体的距离来实现。为了对抗电子转移的指数距离依赖性，这将显著损害基于PeT的传感器响应电压的能力，分子线将用于连接供体与受体。分子线通过将电子转移的机制从近距离处的超交换改变为非常大距离处的电子跳跃来降低电子转移的距离依赖性。分子线将允许有效的电子转移，同时在引入电场时使荧光的变化最大化。传感器可以通过使用化学反应性手柄将探针靶向遗传限定的神经元亚群来进一步改进。将正交化学手柄共价自连接到其自身的膜结合融合蛋白的表达将允许传感器靶向特定的神经元亚型，增加探针响应于电压变化的信噪比，并允许研究异质样品内的神经元的遗传亚型。开发一种电压荧光探针，它可以响应于电压的变化而提供大的荧光增加，同时保持良好的时间保真度并避免电容性负载，这将对研究广泛的神经系统产生广泛的兴趣。 公共卫生关系：光学成像的变化，在神经元细胞的电位提供了一个有吸引力的方法来研究神经元通信的动力学。用于监测神经元活动的传统光学方法响应于跨细胞膜的电压的变化而遭受低信噪比。这项研究将开发新的基于分子线的荧光探针，用于以高空间和时间分辨率光学监测活细胞中的电压。