Engineering Microbial Rhodopsins as Optical Voltage Sensors

描述(申请人提供)：将微生物视紫红素工程作为光学电压传感器神经科学家长期以来一直梦想着一种基因编码的传感器，它可以根据膜电位的变化给出光学信号，目标是对活体神经细胞的电活动进行成像。这种分子还可以用来探测线粒体、心肌细胞、细菌或其他非神经细胞的膜电位，从而为了解与人类健康和疾病有关的广泛细胞的生理状态提供了一个新的窗口。我们建议设计一种荧光跨膜蛋白，其荧光对膜电位敏感。其目标是在活体内可视化单个动作电位。许多团体都试图实现这一目标；我们的做法与以往的努力完全不同。我们的起始材料是一种名为绿色蛋白紫质(GPR)的微生物视紫红质蛋白。在野外，这种蛋白质吸收阳光并泵出质子来产生质子动力。我们将使蛋白质反向运转--使用膜电压来调制光。野生型微生物视紫红质中的视网膜发色团具有足够的荧光，可用于单细胞成像。GPR可在体外培养的斑马鱼神经元和活的斑马鱼神经元中表达和成像。GPR的单点突变导致了一种蛋白质，其荧光对膜电位非常敏感。这个想法的本质是利用膜电位将质子拉向或远离蛋白质中决定颜色的官能团。当细胞处于静止状态时，这个功能基团去质子化，蛋白质变暗。当细胞激发一个动作电位时，一个质子被施加到这个功能基团上，蛋白质变得明亮。就像GFP通过追踪蛋白质在细胞中的位置而彻底改变了生物学一样，我们相信微生物视紫红素将通过它们标记生物膜的能力以及将膜电位转化为荧光变化的能力产生广泛的影响。 与公共卫生相关：许多细胞膜在膜上保持电压差，用于(神经元)的通讯和(细菌和线粒体)能量的产生。我们的目标是开发一种蛋白质，当在细胞中表达时，它可以提供可见的膜电位读数。这种蛋白质将促进对与人类健康和疾病有关的广泛细胞的电生理学的研究。

DESCRIPTION (provided by applicant): Engineering Microbial Rhodopsins as Optical Voltage Sensors Neuroscientists have long dreamed of a genetically encoded sensor that gives an optical signal in response to a change in membrane potential, with the goal of imaging electrical activity of neurons in vivo. Such a molecule could also be used to probe membrane potentials in mitochondria, cardiac cells, bacteria, or in other non-neuronal cells, and thus would provide a new window into the physiological states of a wide range of cells implicated in human health and disease. We propose to engineer a fluorescent transmembrane protein whose fluorescence is sensitive to membrane potential. The goal is to visualize a single action potential in vivo. Many groups have sought to attain this goal; our approach is entirely different from previous efforts. Our starting material is a microbial rhodopsin protein called green proteorhodopsin (GPR). In the wild, this protein absorbs sunlight and pumps protons to generate a proton motive force. We will engineer the protein to run backward-to use membrane voltage to modulate light. The retinal chromophore in wild-type microbial rhodopsins is sufficiently fluorescent for single-cell imaging. GPR can be expressed and imaged in zebra fish neurons in vitro and in living zebra fish. A single-point mutation to GPR leads to a protein whose fluorescence is exquisitely sensitive to membrane potential. The essence of the idea is to use membrane potential to pull a proton toward or away from a color- determining functional group in the protein. When the cell is at rest, this functional group is deprotonated and the protein is dark. When the cell fires an action potential, a proton is forced onto this functional group and the protein becomes bright. Just as GFP revolutionized biology through its ability to track the positions of proteins in cells, we believe that microbial rhodopsins will have a broad impact through their ability to label biological membranes, and to transduce membrane potential into changes in fluorescence. PUBLIC HEALTH RELEVANCE: Many cell membranes maintain a voltage difference across the membrane, which is used for communication (in neurons), and for generation of energy (in bacteria and mitochondria). Our goal is to develop a protein that when expressed in a cell gives a visible readout of the membrane potential. This protein will facilitate studies on the electrophysiology of a wide range of cells implicated in human health and disease.