Development and application of RoCK, a novel Rhodopsin Cyclase/K+ channel-based optogenetic tool for silencing of excitable cells
1) Herwig Baier (PI, Max Planck Institute of Neurobiology, Munich), coworker: Miguel Fernandes
2) Yinth Bernal Sierra (PI; Humboldt University Berlin), coworker:
3) Franziska Schneider (PI; University of Freiburg), coworker: Ramona Kopton
4) R. Seifert
Optogenetics enables manipulation of biological processes with light at unprecedented spatio-temporal resolution to control the behaviour 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 was 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 a number of advantages over other inhibitory tools: i) it is based on a K+ conductance and hence its activation silences cells in the most physiological way without changing the resting membrane potential or disturbing cellular ion gradients, ii) PACK has a propitious photon budget; light pulses of 10 ms at moderate intensity silence action potential generation for > 60 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 of activating other cAMP-signalling pathways.
In the second phase of our SPP project, we will overcome the limitations by developing a new tool using cGMP as a second messenger by employing a rhodopsin cyclase and a small K+ channel (RoCK). We will engineer the rhodopsin cyclase to change its kinetic properties and subsequently be able to modulate RoCK´s time resolution. RoCK functionality will be tested in cardiomyocytes and the nervous system of zebrafish and mice.
Planned Interactions within the project group
R. Seifert: Identification of new as well as engineering and characterization of cGMP-gated K+ channels
Y.A. Bernal Sierra: Engineering of rhodopsin cyclase, characterization of RoCK and application of RoCK in neurons in vitro
F. Schneider-Warme: Applilcation of RoCK to heart cells
H. Baier: Application of RoCK to zebrafish nervous system in vivo
Publications Baier, relevant for the proposal
Barker A, Baier H (2015). Sensorimotor decision-making in the zebrafish tectum. Curr Biol 25:2804-14.
Kubo F, Hablitzel B, Dal Maschio M, Driever W, Baier H, Arrenberg AB (2014). Functional architecture of an optic flow responsive area that drives horizontal eye movements in zebrafish. Neuron 81:1344-59.
Thiele T, Donovan JC, Baier H (2014). Modular descending control of swim posture in zebrafish. Neuron 83: 679-691.
Arrenberg AB, Stainier DY, Baier H, Huisken J (2010). Optogenetic control of cardiac function. Science 330:971-974.
Arrenberg AB, Del Bene F, Baier H (2009). Optical control of zebrafish behavior with halorhodopsin. Proc Natl Acad Sci U S A 106:17968-17973.
Publications Schneider-Warme, relevant for the proposal
B. R. Rost*, F. Schneider*, M. K. Grauel, C. Wozny, C. Bentz, A. Blessing, T. Rosenmund, T. Jentsch, D. Schmitz, P. Hegemann, and C. Rosenmund, “Optogenetic Acidification of Synaptic Vesicles and Lysosomes,” Nature Neuroscience, advance online publication 09 Nov 2015, doi 10.1038/nn.4161
F. Schneider, C. Grimm and P. Hegemann, “The biophysics of channelrhodopsin”, Annual Review of Biophysics, vol. 44, pp. 167-186, 2015
J. Wietek, J. S. Wiegert, N. Adeishvili, F. Schneider, H. Watanabe, S. P. Tsunoda, A. Vogt, M. Elstner, T. G. Oertner, and P. Hegemann, “Conversion of Channelrhodopsin into a Light-Gated Chloride Channel,” Science, Vol. 344, no. 6182, pp. 409-412, 2014.
F. Schneider, D. Gradmann, and P. Hegemann, “Ion Selectivity and Competition in Channelrhodopsins,” Biophysical Journal, vol. 105, pp. 91-100, July 2013.
M. Prigge*, F. Schneider*, S. P. Tsunoda, C. Shilyansky, J. Wietek, K. Deisseroth, and P. Hegemann, “Color-tuned Channelrhodopsins for Multiwavelength Optogenetics.,” The Journal of Biological Chemistry, 2012. * equal contribution