Ultrafast Genetically Encoded Voltage Indicators Designed from First Principles

摘要： 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的PAR分辨率将允许生理学的关联 从单个细胞和细胞类型，到作为行为、认知和 在正常和患病的大脑中观察到的情感状态。遗传编码电压指示器(GEVI) 作为细胞类型的特定探针，如果它们的电压敏感度、亮度和 时间分辨率可以被设计成提供对高频动作电位的可靠检测， 亚阈值“迷你”的检测对突触伸缩和内稳态可塑性至关重要，并能够 解析有助于推断特定通道/受体对尖峰和突触的贡献的波形 变速箱。我们建议通过合理的设计来发明下一代GEVI，从非第一原理出发 生物衍生的蛋白质。我们将采用人造蛋白质“模型”，这些模型是全新设计的，是刚性的。 4-螺旋束蛋白质，作为定制支架，在细胞内任意定位生物辅助因子 脚手架核心。胆绿素发色团在跨膜模型中的战略定位允许 光学斯塔克效应的电压传感，其中生色团表现出电场诱导的电压变化 导致观测到的荧光发生超快变化的吸光度。我们称这些蛋白质为“主体” (Maquette Stark Effect记者)超快红外荧光记者将概括整个细胞 没有明显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.