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回路中信号处理的能力，并有望为治疗不同疾病和残疾提供新的方法。光遗传学的核心工具是驱动视网膜膜蛋白。不幸的是，目前只有少数视网膜蛋白可用于光遗传学，用于去极化从而激活神经元的非选择性通道视紫红质ChR2， Cl-泵盐紫红质NphR和H+泵古视紫红质Arch3，两者都用于超极化从而沉默神经元。识别和/或设计具有新特性的光驱动蛋白，例如对特定离子具有高选择性和导电性的通道和转运体，将对未来的进展至关重要。我们的项目分为两个互补的部分，解决光调节泵(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确定/产生新的光驱动泵。我们最近以高分辨率解决了第一个已知的光驱动Na+泵，Krokinobacter eikastus rhodopsin 2 （KR2）的基态结构。离子转运途径的确定使光驱动K+泵的设计成为可能。我们将优化KR2和K+泵在神经元中的表达，以实现广泛的光遗传学应用。此外，我们将从基因组数据库中选择新的潜在候选者，以获得具有新特性的Na+泵，并通过修改这些选择的Na+泵来设计额外的K+泵。为了理解离子泵送的分子机制，我们将解决KR2的中间态结构和相关的K+泵送。这种结构信息将用于改善Na+和K+泵的输运，并进一步用于工程光驱动Ca2+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这种新型泵，从哺乳动物细胞系到秀丽隐杆线虫。(2)以往试图改变ChR2阳离子选择性的尝试收效甚微。其中一个原因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，并为设计具有更高选择性和传导性能的光激活离子通道提供线索。由三个法国和三个德国团队组成的联盟包括研究光驱动蛋白质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技术，从视网膜蛋白生产、功能和结构测定、生物物理表征、合理蛋白设计到光驱动蛋白在神经科学、神经元细胞培养和线虫中的应用。我们的目标是提出并实现一套新的光驱动蛋白，以大大推进光遗传学。

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.