Constructing of genetically encoded light-activated K+-channels with fast gating and distinct trafficking properties

对于许多光遗传学应用，有必要使靶细胞的膜过度活化以终止例如神经元的放电。目前这是通过基于视蛋白的泵或光敏阴离子通道实现的。然而，这些蛋白质具有一些缺点，并且它们的应用限于特定类型的神经元（阴离子通道）或引起细胞中的异常离子梯度（泵）。由于质膜的超极化通常在细胞中由K+选择性通道产生，因此对光敏K+通道作为光遗传学工具的需求很大。用于此目的的理想通道将是完全遗传编码的K+选择性通道，具有大的单位电导，其以可逆的方式快速响应暗/光转换，并且被有效地分选到质膜或另一选择的内膜。为此目的而制造的合成光敏K+通道并不满足所有这些标准。因此，本提案的目标是工程化具有合适性质的完全遗传编码的光敏通道，用于光遗传学中的广泛和稳健的应用。该项目将建立在光敏K+通道的原型上，该原型是通过将来自Avena sativa的蓝光传感器LOV 2的一部分与一个小的病毒K+通道融合而成功构建的。该项目的一部分将改善该通道的表达和质膜分选，并优化神经元的这些标准。这将通过增加有效的分选信号、优化固有的分选动机和定向进化策略来实现。由于原型通道对暗/亮转换的响应缓慢，因此该项目的第二部分将改善通道激活/去激活响应光的动态。这将通过光敏通道设计中的新原理来实现。为此，我们将用小的miniSOG结构域或感觉性视紫红质代替慢LOV结构域作为光传感器。在miniSOG的情况下，蓝光应该引起单线态氧的快速生成，然后门控附着的氧化还原敏感通道。在另一种设计中，光诱导的感觉视紫红质构象变化应该机械地传递到通道门。任何有希望的候选通道的功能特性将通过电生理记录来表征，并移交给财团中的其他小组进行体内系统测试。

For many optogenetic applications it is necessary to hyperpolarize the membrane of target cells in order to terminate for example firing of neurons. This is currently achieved by opsin-based pumps or light-sensitive anion channels. These proteins however have some disadvantages and their application is limited to specific types of neurons (anion channels) or cause abnormal ion gradients in cells (pumps). Since a hyperpolarization of the plasma membrane is typically generated in cells by K+-selective channels, there is a great demand for light-sensitive K+ channels as optogenetic tools. Ideal channels for this purpose would be fully genetically encoded, K+-selective channels, with a large unitary conductance, which respond rapidly to dark/light transitions in a reversible fashion and which are efficiently sorted to the plasma membrane or another endomembrane of choice. Synthetic light-sensitive K+-channels, which were made for this purpose, do not fulfill all these criteria. The goal of the present proposal is therefore the engineering of a fully genetically encoded light-sensitive channel with suitable properties for a wide and robust application in optogenetics. The project will build on a prototype of a light-sensitive K+-channel, which was successfully build by fusing parts of the blue-light senor LOV2 from Avena sativa with a small viral K+-channel. One part of the project will improve expression and plasma membrane sorting of this channel and optimize these criteria for neurons. This will be achieved by an addition of efficient sorting signals, optimization of inherent sorting motives and directed evolution strategies. Since the prototype channel suffers from a slow response to dark/light transitions the second part of the project will improve the dynamics of channel activation/deactivation in response to light. This will be achieved by novel principles in the design of light-sensitive channels. For this purpose we will replace the slow LOV domain as light sensor either by the small miniSOG domain or by sensory rhodopsins. In the case of the miniSOG blue light should cause a rapid generation of singlet oxygen, which then gates an attached redox-sensitive channel. In the alternative design the light-induced conformational change of sensory rhodopsins should be mechanically transmitted to channel gates. The functional properties of any promising candidate channel will be characterized by electrophysiological recordings and handed over to other groups in the consortium for testing in in vivo systems.