Ultrafast Genetically Encoded Voltage Indicators Designed from First Principles

摘要： 利用时间和精细特征波形光学记录神经元电活动的能力 与全细胞膜片钳电生理学相当的分辨率将允许生理学的相关性， 个体细胞和细胞类型的神经回路水平活动，这些活动是行为，认知和 在正常和患病大脑中观察到的情感状态。基因编码电压指示器（GEVI） 作为细胞类型特异性探针， 可以设计时间分辨率以提供高频动作电位的可靠检测， 检测对突触缩放和稳态可塑性至关重要的亚阈值“minis”，以及 解析波形，用于推断特定通道/受体对尖峰和突触的贡献 传输我们建议发明下一代GEVI通过合理的设计，从第一原则的非- 生物衍生蛋白质。我们将采用人工蛋白质“maquettes”，这是新设计的和刚性的 4-螺旋束蛋白，其用作定制支架，用于任意地将生物辅因子定位在 脚手架核心胆绿素发色团在跨膜模型内的战略定位允许 通过光学斯塔克效应的电压传感，其中发色团表现出电场诱导的变化， 这导致观察到的荧光的超快变化。我们称这些蛋白质为“MASTER” （Maquette Stark Effect）超快红外荧光报告将重演全细胞 没有可观察到的延迟或波形差异的记录。

ABSTRACT: The ability to optically record neuronal electrical activity with the temporal and fine-feature waveform resolution on par with whole-cell patch clamp electrophysiology would permit the correlation, of the physiology of individual cells and cell types, to the neural circuit-level activity that underlies behaviors, cognition, and affective states observed in the normal and diseased brain. Genetically encoded voltage indicators (GEVIs) hold great promise for this purpose as cell-type specific probes, if their voltage-sensitivity, brightness, and temporal resolution can be engineered to provide the reliable detection of high frequency action potentials, the detection of sub-threshold “minis” critical to synaptic scaling and homeostatic plasticity, and the ability to resolve waveforms useful for deducing specific channel/receptor contributions to spiking and synaptic transmission. We propose to invent next-generation GEVIs through rational design from first principles of non- biologically derived proteins. We will adapt artificial protein “maquettes,” which are de novo-designed and rigid 4-helix bundle proteins that serve as custom scaffolds for arbitrarily positioning biological co-factors within the scaffold core. Strategic positioning of a biliverdin chromphore within a transmembrane maquette allows for voltage sensing by the optical Stark effect, in which chromophores exhibit electric field-induced changes in absorbance that result in ultrafast changes in observed fluorescence. We call these proteins, “MASTERs” (Maquette Stark Effect Reporters). The ultrafast infrared-fluorescent reporters will recapitulate whole-cell recordings with no observable delay or waveform difference.