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.
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