New Light-Driven Channels and Transporters for Optogenetics

光遗传学正在彻底改变我们研究神经元电路中信号处理的能力，并有望为治疗不同的疾病和障碍提供新的方法。光遗传学的核心工具是光驱动的视网膜膜蛋白。不幸的是，目前用于光遗传学的视网膜蛋白很少，用于去极化并激活神经元的非选择性通道视紫红质ChR2，氯泵卤视紫红质NphR和H泵古紫红质Arch3，这两种蛋白都用于超极化从而使神经元沉默。识别和/或设计具有新特性的光驱动蛋白质，如对特定离子具有高选择性和导电性的通道和转运体，将是未来进展的关键。我们的项目分为两个互补的部分，分别涉及光调节泵(1)和光调节通道(2)。(1)我们将根据我们联盟成员最近的工作，识别/产生新的光驱动泵。我们最近在高分辨率下解决了已知的第一个光驱动钠泵--Krokinbacter eikastus视紫红质2(KR2)的基态结构。离子转移途径的确定使设计出一种光驱动的钾泵成为可能。我们将优化KR2和工程K泵在神经元中的表达，以便广泛应用于光遗传。此外，我们将从基因组数据库中选择新的候选基因，以获得具有新特性的钠泵，并通过对这些选择的钠泵进行改造来设计更多的K泵。为了了解离子泵浦的分子机制，我们将解出KR2中间态的结构以及与之相关的K泵。这些结构信息将被用于改善钠泵和钾泵的运输，并进一步用于设计光驱动的钙泵。为了评估光遗传学的潜力，我们将在多种表达系统中表达新的泵，从哺乳动物细胞系到线虫。(2)以前对ChR2阳离子选择性进行修饰的尝试只取得了有限的成功。原因之一是离子渗透的分子机制还不够清楚。我们将结合结构生物学和计算生物学来描述渗透和选择性过程，并为设计具有更好的选择性和导电性的光激活离子通道提供线索。这个由三个法国团队和三个德国团队组成的联盟包括光驱动蛋白质研究的先驱和光遗传学的创始人。其互补的多学科专业知识涵盖了所有可用的技术，从视网膜蛋白质生产、功能和结构确定、生物物理表征、合理的蛋白质设计到将光驱动蛋白质应用于神经科学、神经元细胞培养和线虫线虫。我们的雄心是建议和实施一套新的光驱动蛋白质，这是大幅推进光遗传学所需的。

Optogenetics is revolutionizing our ability to study signal processing in neuronal circuits and promises new approaches to the treatment of different diseases and handicaps. The core tools of optogenetics are light-driven retinal membrane proteins. Unfortunately, there are at present only few retinal proteins available for optogenetics, the non-selective channel rhodopsin ChR2 used to depolarize and thus activate neurons, the Cl- pump halorhodopsin NphR, and the H+ pump archaerhodopsin Arch3, both of which are used to hyperpolarize and thus silence neurons. Identification and/or engineering light-driven proteins with novel properties, such as channels and transporters with high selectivity and conductivity for a particular ion will be crucial for future progress. Our project is divided in two complementary parts addressing light-regulated pumps (1) and light-regulated channels (2).(1) We will identify/generate new light-driven pumps, based on recent work of members of our consortium. We recently solved the ground state structure of the first known light-driven Na+ pump, Krokinobacter eikastus rhodopsin 2 (KR2), at high resolution. The identification of the ion-translocation pathway allowed engineering a light-driven K+ pump. We will optimize expression of KR2 and the engineered K+ pump in neurons for widespread optogenetic use. Moreover, we will select new prospective candidates from genome databanks to obtain Na+ pumps with novel properties and engineer additional K+ pumps by modifying these selected Na+ pumps. To understand the molecular mechanism of ion pumping, we will solve the structures of intermediate states of KR2 and the related K+ pump. This structural information will be used for improved transport by Na+ and K+ pumps and furthermore for engineering light-driven Ca2+ pumps. To assess the potential for optogenetics, we will express the novel pumps in multiple expressions systems, ranging from mammalian cell lines to C. elegans. (2) Previous attempts to modify the cationic selectivity of ChR2 have met limited success. One reason is that the molecular mechanism of ion permeation is insufficiently clear. We will combine structural biology and computational biology to describe the permeation and selectivity process, and to obtain clues toward the design of light-activated ion channels with improved selectivity and conduction properties. The consortium of three French and three German teams includes pioneers of the studies of light-driven proteins and founders of optogenetics. Its complementary multidisciplinary expertise spans the full range of available techniques, from retinal protein production, function and structure determination, biophysical characterization, rational protein design to the application of light-driven proteins to neuroscience, in neuronal cell culture and in the nematode C. elegans. Our ambition is to suggest and implement a set of new light-driven proteins required to substantially advance optogenetics.