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，目前只有少数视网膜蛋白可用于光遗传学，非选择性通道视紫红质ChR 2用于去极化并因此激活神经元，Cl-泵盐视紫红质NphR和H+泵古视紫红质Arch 3，这两者都用于过度极化并因此沉默神经元。识别和/或工程化具有新特性的光驱动蛋白质，例如对特定离子具有高选择性和导电性的通道和转运蛋白，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。我们最近解决了基态结构的第一个已知的光驱动的Na+泵，Krokinaseikastus视紫红质2（KR 2），在高分辨率。离子易位途径的鉴定允许设计光驱动的K+泵。我们将优化神经元中KR 2和工程K+泵的表达，以供广泛的光遗传学用途。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，并通过修改这些选定的Na+泵来设计额外的K+泵。为了理解离子泵的分子机制，我们将解决KR 2和相关的K+泵的中间态的结构。这种结构信息将用于改善Na+和K+泵的运输，并进一步用于工程光驱动的Ca 2+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C。优雅(2)以前尝试修改ChR 2的阳离子选择性，取得了有限的成功。原因之一是离子渗透的分子机制不够清楚。我们将结合联合收割机和计算生物学来描述渗透和选择性的过程，并获得线索的光激活离子通道的设计与改善的选择性和传导性能。这个由三个法国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，功能和结构测定，生物物理表征，合理的蛋白质设计到光驱动蛋白质在神经科学中的应用，在神经元细胞培养和线虫C。优雅我们的目标是提出并实施一套新的光驱动蛋白质，以大幅推进光遗传学。

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.