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

对于许多光遗传应用来说，为了终止例如神经元的放电，必须使靶细胞的膜超极化。这目前是通过基于光学蛋白的泵或光敏阴离子通道来实现的。然而，这些蛋白质有一些缺点，它们的应用仅限于特定类型的神经元(阴离子通道)或导致细胞中异常的离子梯度(泵)。由于细胞质膜的超极化通常是由K+选择性通道产生的，因此对光敏K+通道作为光遗传工具的需求很大。为此，理想的通道应该是完全遗传编码的K+选择性通道，具有大的单位电导，以可逆的方式对暗/光转换做出快速反应，并有效地分选到质膜或另一种选择的内膜上。为此目的而制造的合成光敏K+通道并不符合所有这些标准。因此，本提案的目标是设计一种完全遗传编码的光敏通道，该通道具有在光遗传学中广泛而稳健的应用的适当特性。该项目将建立一个光敏K+通道的原型，该原型是通过将来自燕麦的部分蓝光感受器LOV2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类，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/光转换的反应迟缓，该项目的第二部分将改进通道对光的激活/去激活的动力学。这将通过设计光敏通道的新原理来实现。为此，我们将用小的微型SOG结构域或感觉性视紫红质取代慢LOV结构域作为光传感器。在微型SOG的情况下，蓝色光应该会导致单线态氧的快速生成，然后门控连接的氧化还原敏感通道。在替代设计中，感觉性视紫红质的光诱导构象变化应该被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，并移交给该联盟中的其他小组在体内系统中进行测试。

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.