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

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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.