Improved Optogenetic Tools: Characterization of new rhodopsins and engineering of novel features into existing photoreceptors
For the high resolution of cellular receptor dynamic, optogenetic methods are ideal. The non-invasive and reversible stimulation of cells by visible light provides an advantage to pharmacological or electrophysiological intervention. Optogenetics is a rapidly evolving field with a still increasing number of applications and of laboratories developing new tools or using the established ones. Modulation (“activation” or “inhibition”) of cellular activity by visible light via genetically encoded proteins is an elegant, non-invasive complementary approach to the very successful imaging of cellular function via GFP and related engineered proteins (“optogenetic imaging”). As the specific tasks for modulation of cells are widening, the performance requirements of optogenetic actuators also increase.
The breakthrough for “optogenetic actuators” came with the discovery of channelrhodopsin-2, a directly light-gated cation channel, able to depolarize cells and to light-modulate behavior of transgene animals. The success of channelrhodopsin-2 encouraged us to introduce halorhodopsin as a hyperpolarizing and “neuron-inhibiting” optogenetic protein. Halorhodopsin, as well as Channelrhodopsin, however, have certain limitations due to their relatively small charge transfer per protein molecule and their requirement for high light intensity and high protein expression. Recently we introduced a channelrhodopsin-2 mutant with improved expression and dramatically increased photocurrents, even at low light intensity. In this project we plan to develop further new and improved optogenetic actuators with larger charge transfer per molecule and lower light intensity requirement, based on engineering of established optogenetic tools and characterization of new photoreceptors.
We plan to apply the recently by us identified mutations, which increase the expression of channelrhodopsin-2 [Dawydow et al. (2013) Proc Natl Acad Sci 1211, 13972-7], to the 2014 published chloride-permeable channelrhodopsin variants [Berndt et al. (2014) Science 344, 420-5]. We also plan to couple cyclic nucleotide-gated (CNG) cation or CNG potassium (CNGK) channels to light-activated adenylyl or guanylyl cyclases. Compared to channelrhodopsin these channels show higher single channel conductance. Once made light-sensitive, then even sparse expression (compared to channelrhodopsin and halorhodopsin) will allow light-activated de- (CNG) or hyper-(CNGK) polarization. To this aim we will work with different photoactivated cyclases which we characterized in our laboratory in recent years [Schröder-Lang et al. (2007) Nat Methods 4, 39-42; Stierl et al. (2011) J Biol Chem 286, 1181-8; Raffelberg et al. (2013) Biochem J 455, 359-65]
Georg Nagel will coordinate and supervise the project together with other members of the team.
|A LOV domain-mediated, blue light-activated Adenylyl Cyclase from the cyanobacterium Microcoleus chthonoplastes PCC 7420||2013||Raffelberg, S., Wang, L., Gao, S., Losi, A., Gärtner, W., Nagel, G.||Biochem J||More|
|Degradation of channelopsin-2 in the absence of retinal and degradation resistance in certain mutants||2013||Ullrich, S., Gueta, R., Nagel, G.||Biol Chem||More|
|Light modulation of cellular cAMP by a small bacterial photoactivated adenylyl cyclase, bPAC, of the soil bacterium Beggiatoa||2011||Stierl, M., Stumpf, P., Udwari, D., Gueta, R., Hagedorn, R., Losi, A., Gärtner, W., Petereit, L., Efetova, M., Schwärzel, M., Oertner, T.G., Nagel, G., Hegemann, P.||J Biol Chem||More|
|Fast manipulation of cellular cAMP level by light in vivo||2007||Schröder-Lang, S., Schwärzel, M., Seifert, R., Strünker, T., Kateriya, S., Looser, J., Watanabe, M., Kaupp, J.F., Hegemann, P., Nagel, G.||Nat Methods||More|
|Multimodal fast optical interrogation of neural circuitry||2007||Zhang, F., Wang, L.-P., Brauner, M., Liewald, J.F., Kay, K., Watzke N., Wood, P.G., Bamberg, E. Nagel, G., Gottschalk, A., Deisseroth, K.||Nature||More|
|Channelrhodopsins: directly light-gated cation channels||2005||Nagel, G., Szellas, T., Kateriya, S., Adeishvili, N, Hegemann, P., Bamberg, E.||Biochem Soc Trans||More|
|Light activation of channelrhodopsin-2 in excitable cells of Caenorhabditis elegans triggers rapid behavioral responses||2005||Nagel, G., Brauner, M., Liewald, J.F., Adeishvili, N., Bamberg, E., Gottschalk, A.||Curr Biol||More|
|Channelrhodopsin-2, a directly light-gated cation-selective membrane channel||2003||Nagel, G, Szellas, T., Huhn, W., Kateriya, S., Adeishvili, N., Berthold, P., Ollig, D., Hegemann, P., Bamberg, E.||Proc Natl Acad Sci USA||More|
|Channelrhodopsin-1: a light-gated proton channel in green algae||2002||Nagel, G., Ollig, D., Fuhrmann, M., Kateriya, S., Musti, A.M., Bamberg, E., Hegemann, P.||Science||More|