Potassium channel-based optogenetic tool development - Establishment and optimization of compartment-specific light-inducible silencing tools

Optogenetics offers unique possibilities to control cells with a high spatio-temporal precision using light-sensitive proteins. There are two groups of light-inducible proteins: the first comprises retinal-containing proteins, which are light-inducible ion pumps or channels. The second group includes modular photoactuators, in which light induces conformational changes which in turn cause interaction with an effector domain. Optogenetics is most widely used to study excitable cells and has the potential to serve as a treatment for a lot of diseases like neurological disorders or blindness due to degradation of photoreceptors. In this work, potassium channel-based optogenetic tools were developed, optimized and functionally characterized. The goal was to target the respective GFP-tagged channels to two distinct locations within the cell: (i) the plasma membrane, where potassium channels can serve as optogenetic silencing tools by inducing hyperpolarization, (ii) the inner mitochondrial membrane, where light-inducible depolarization by potassium channel activity offers the chance to study the so far unknown function of potassium channels in mitochondria. To achieve targeting to the plasma membrane, two candidate channels were examined: KcvPBCV-1 and KcvNTS. Kesv and Kmpv12T on the other hand served as candidate channels to study the effects of potassium channel expression in the inner mitochondrial membrane. To allow a most effective sorting of these four candidate channels, two different approaches were pursued to optimize the sorting efficiency of these channels in cultured mammalian cells. One effective way of augmenting sorting of channels to the mitochondria was achieved by codon optimization. For this purpose, rare codons in the channel coding gene were replaced by frequently used codons. This procedure resulted, in particular in Kesv, in an enhanced sorting to the mitochondria. Another effect which markedly increased sorting efficiency to the mitochondria was to shorten the protein linker between the channel protein and its GFP-tag. The combination of these two steps, a codon optimized channel combined with a short protein linker, yielded a very efficient and exclusive sorting of Kesv to the mitochondria. This positive effect of codon optimization on mitochondrial targeting was reproducible in six different cell lines, suggesting that this is a general mechanism in mammalian cells. KcvPBCV-1, which is by default sorted to the secretary pathway and further to the plasma membrane, was insensitive to codon optimization. In contrast to the mitochondrial potassium channels, the two channels sorted to the plasma membrane favor low expression levels to achieve the most effective sorting. To design an optogenetic tool for short-term depolarization of the mitochondria, the existing light-gated potassium channel BLINK1 was reengineered for mitochondrial sorting. It occurred that the addition of six canonical mitochondrial targeting sequences to the N-terminus of the protein resulted in an effective sorting of this protein to the mitochondria. Its functionality in the mitochondria remains to be tested. In a different strategy, optogenetic tools for long-term activation of potassium channels in the plasma membrane or the mitochondria were developed. In this case, two different light-inducible gene expression systems were employed to regulate expression of suitable potassium channels by light. The first approach was based on dimerization of the bacterial light-oxygen-voltage domain from Erythrobacter litoralis (EL222). The second system was based on the light-regulated interaction of cryptochrome 2 (CRY2) with its interacting partner CIB1. In these experiments the expression of a GFP-tagged potassium channel was placed under control of the EL222 system. Based on the intensity of the GFP signal in transfected cells, it became clear that the channel of interest was already significantly expressed in the dark and required a very high light intensity (120 µE for 16 hours) for elevating expression above the dark level. Because of this combination of unspecific leak expression in the dark and high light requirement, the EL222 system was not further pursued. In an alternative system, potassium channel expression was controlled by the blue light receptor system CRY2/CIB1 from Arabidopsis thaliana. This light-sensitive transcription system proved to be effective for the purpose of light-inducible potassium channel expression. It required very low light intensities (6 µE for 1 hour) or short exposure times for a robust expression of the candidate channel. A 5 second pulse of 120 µE was already sufficient for a maximal stimulation of channel expression. Leak expression of the channel in the dark was barely above detection limit. It was also possible to simplify the handling of the system by placing all three necessary components (CRY2, CIB1, potassium channel) in one expression vector without affecting its efficacy. We found that one hour of pulsed light (6 µE) was sufficient to achieve full activation one hour after light-stimulation. The CRY2 CIB1 system was examined in different functional assays. The properties of channels which were expressed at the plasma membrane in a light-dependent manner, were examined by whole cell patch clamp measurements. The data show that the channels, which were expressed under control of the CRY2 CIB1 system reached the plasma membrane and there exhibited the same functional properties as the constitutively expressed control channels. The functionality of channels expressed in the inner mitochondrial membrane, were examined in planar lipid bilayer experiments as well as by a set of microscopy assays. By using fluorescent reporters of the mitochondrial membrane voltage and mitochondrial calcium, it occurs that expression of mitochondrially targeted potassium channels under control of the CRY2 CIB1 system caused an effective depolarization of theses organelles. Also, they did significantly lower the mitochondrial calcium levels, but did not induce apoptosis. Currently, the CRY2 CIB1 constructs undergo functional tests in vivo to examine whether they influence the development of zebrafish larvae if expressed in different organ systems