Development and application of RoCK, a novel Rhodopsin Cyclase/ K+ channel-based optogenetic tool for silencing of excitable cells

RoCK的开发和应用，一种新型的基于视紫红质环化酶/K通道的光遗传学工具，用于沉默可兴奋细胞

• 批准号: 315193289
• 负责人: Professor Dr. Herwig Baier
• 金额: --
• 依托单位: Max-Planck-Institut für Neurobiologie des Verhaltens - caesar
• 依托单位国家: 德国
• 项目类别: Priority Programmes
• 财政年份: 2016
• 资助国家: 德国
• 起止时间: 2015-12-31 至 2022-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/315193289?language=en
• 关键词: Development; application; RoCK; novel; Rhodopsin

Optogenetics enables manipulation of biological processes with light at unprecendented spatio-temporal resolution to control the behavior of cells, networks, or even whole animals. In contrast to the performance of excitatory rhodopsins, the effectiveness of inhibitory optogenetic tools is still insufficient. In order to overcome the limitations and side effects imposed by the currently available silencing tools, we developed a K+ based optogenetic tool (PACK) in the first phase of our SPP1926 project. PACK is a two-component system composed of a light-activated adenylyl cyclase PAC and the cAMP-gated channel SthK, driving large K+ currents upon short blue-light pulses in the target cells. PACK is able to inhibit action potentials in neurons of mice and zebrafish in vitro and in vivo, as well as to silence excitation and contraction of rabbit cardiomyocytes. PACK has two main advantages over other inhibitory tools: i) it is based on a K+ conductance and hence its activation silences cells in the most natural way without changing the resting membrane potential or disturbing cellular ion gradients, and ii) PACK has a propitious photon budget; light pulses of 10 ms at moderate intensity silence action potential generation for tens of seconds. However, the use of PACK is limited by certain drawbacks such as slow off-kinetics (caused by the intrinsically slow photocycle kinetics of PAC), and possible side effects (by activating other cAMP-signaling pathways). In the second phase of our SPP project, we will overcome these limitations by using cGMP as a second messenger, employing a rhodopsin cyclase and a small K+ channel (RoCK). In preliminary experiments with the recently characterized rhodopsin guanylyl cyclase from the Catenaria anguillulae (CaRhGC) and a novel engineered “prototype” mutant SthK channel, expressed in the neuroblastoma/DRG hybrid cell line (ND7/23) and in the cardiomyocyte cell line (HL-1), short light-pulses triggered large outward K+ currents of similar size but with an order of magnitude faster off-kinetics compared to PACK. We aim to develop RoCK as a novel inhibitory tool by selecting and/or engineering optimized versions of the guanylyl cyclase and the K+ channel. We will generate RoCK variants with red-shifted action spectrum, and versions targeted to presynaptic terminals. RoCK constructs will be initially tested in vitro, using cell lines, primary hippocampal neurons and isolated cardiomyocytes, before applying them to optically mimic atrial ablation lines in isolated rabbit hearts in situ and to modulating behavioral responses in zebrafish in vivo. We expect that RoCK will be a versatile addition to the optogenetic toolbox, with many applications in neurosciences, cardiac research, and beyond.

光遗传学能够以前所未有的时空分辨率利用光操纵生物过程，以控制细胞、网络甚至整个动物的行为。与兴奋性视紫红质的表现相比，抑制性光遗传工具的有效性仍然不足。为了克服目前可用的沉默工具的局限性和副作用，我们在SPP1926项目的第一阶段开发了一个基于K+的光遗传工具(PACK)。PACK是一个由光激活的腺苷环化酶PAC和cAMP门控通道SthK组成的双组分系统，在靶细胞中用短的蓝光脉冲驱动大的K+电流。Pack在体内外均能抑制小鼠和斑马鱼神经元的动作电位，并能抑制兔心肌细胞的兴奋和收缩。与其他抑制工具相比，PACK有两个主要优势：i)它基于K+电导，因此它的激活以最自然的方式使细胞沉默，而不会改变静息膜电位或干扰细胞离子梯度；ii)PACK具有有利的光子预算；10ms的中等强度光脉冲静默动作电位可产生数十秒。然而，Pack的使用受到某些缺点的限制，如缓慢的关闭动力学(由PAC固有的缓慢光循环动力学引起)和可能的副作用(通过激活其他cAMP信号通路)。在SPP项目的第二阶段，我们将使用cGMP作为第二信使，利用视紫红质环化酶和小K+通道(ROCK)来克服这些限制。在对最近鉴定的鳗链视紫红质鸟酰环化酶(CaRhGC)和在神经母细胞瘤/DRG杂交细胞系(ND7/23)和心肌细胞系(HL-1)中表达的新型工程“原型”突变SthK通道的初步实验中，短光脉冲触发了大的外向K+电流，其大小类似，但非动力学特性比PACK快一个数量级。我们的目标是通过选择和/或设计鸟苷酸环化酶和K+通道的优化版本来开发ROCK作为一种新的抑制工具。我们将生成具有红移动作频谱的摇滚变体，以及针对突触前终端的版本。ROCK构建物将首先在体外进行测试，使用细胞系、原代海马神经元和分离的心肌细胞，然后将它们应用于光学模拟离体兔心脏的心房消融线，并在体内调节斑马鱼的行为反应。我们预计ROCK将成为光遗传工具箱的一个多功能补充，在神经科学、心脏研究等领域有许多应用。