Tuning wavelength and G protein specificity of Melanopsin for optogenetic control of G protein signaling pathways.
Participants:
1) Klaus Gerwert (PI; University of Bochum), coworker:
2) Stefan Herlitze (PI; University of Bochum), coworker:
3) Tobias Brügmann (PI; University of Göttingen), coworker:
Tuning wavelength and G protein specificity of bistable Melanopsin for optogenetic control of G protein signaling pathways.
Our goal is to create optogenetic tools for independent control of intracellular G protein signals activated by the Gi/o and Gs, but in particular the Gq/11 pathway. These tools will be established using vertebrate melanopsin and neuropsin as light-activated G protein coupled receptor (GPCR), which activates Gq11 and Gi/o pathways in neurons, heart and heterologous expression systems. Computer models (Gerwert lab) will predict amino acid position critical for wavelength specificity, bistabililty, and G protein selectivity. The functional analysis (Brügmann & Herlitze lab) of amino acid changes at predicted position using electrophysiological recordings and imaging techniques will provide experimental feedback for new working models of melanopsin and neuropsin and their G protein selectivity. Vertebrate melanopsin and neuropsin as bistable pigments are preferable over commonly used opsins to control G protein pathways in particular for highly-repetitive in vivo applications, because it can be switched on and off by two different wavelength of visible light. In addition, sustained G protein signals can be activated by short light pulses, reducing phototoxicity. These tools will be applicable for controlling every GPCR coupling to the common G protein pathways, such as dopamine, adreno, metabotropic glutamate, histamine or orexin receptor pathways without change in signal kinetics. Because of our long-standing interest in serotonin, we will tailor these tools to specifically control G protein signals in 5HT receptor signaling domains. The ultimate goal is to control two signaling pathways simultaneously, but independently by two different wavelength of light to understand how G protein signals synergistically and/or independently act to modulate cell function and behavior.
Logic of interaction:
In order to perform these experiments we have assembled a team of three experts on the development of optogenetic tools (Brügmann & Herlitze) and computational modeling of protein structures (Gerwert).
Publications Herlitze:
Masseck, O.A., Spoida, K., Dalkara, D., Maejima, T., Rubelowski, J.M., Wallhorn, L., Deneris, E.S. and Herlitze, S. (2014) Vertebrate cone opsins enable sustained and highly sensitive rapid control of Gi/o signaling in anxiety circuitry. Neuron 19(81): 1263-73.
Spoida, K., Masseck, O.A., Deneris, E.S. and Herlitze, S. (2014). Gq/5-HT2c receptor signals activate a local GABAergic inhibitory feedback circuit to modulate serotonergic firing and anxiety in mice. Proc. Natl. Acad. Sci., USA, 111(17):6479-84.
Gutierrez, D.V., Mark, M.D., Masseck, O., Maejima, T., Kuckelsberg, D., Hyde, R.A., Krause, M., Kruse, W., Herlitze, S. (2011). Optogenetic Control of Motor Coordination by Gi/o Protein-coupled Vertebrate Rhodopsin in Cerebellar Purkinje Cells. J. Biol. Chem., 286, 25848-58.
Oh, E., Maejima, T., Liu, C., Deneris, E.S., Herlitze, S. (2010). Substitution of 5-HT1A receptor signaling by a light-activated G protein-coupled receptor. J. Biol. Chem., 85, 30825-36.
Li, X., Gutierrez, D., Hanson, G., Han, J., Mark, M.D., Chiel, H., Hegemann, P., Landmesser, L.T., Herlitze, S. (2005). Fast non-invasive control of neuronal excitability and network behavior by vertebrate rhodopsin and green algae channelrhodopsin. Proc. Natl. Acad. Sci., USA, 102, 17816-17821.
Publications Gerwert:
Wolf, S., Böckmann, M., Höweler, U., Schlitter, J. & Gerwert, K. (2008) Simulations of a G protein-coupled receptor homology model predict dynamic features and a ligand binding site. FEBS Letters 582, 3335–3342.
Wolf, S., Freier, E., Potschies, M., Hofmann, E. & Gerwert, K. (2010) Directional Proton Transfer in Membrane Proteins Achieved through Protonated Protein-Bound Water Molecules: A Proton Diode. Angew. Chem. Int. Ed. 49, 6889–6893.
Freier, E., Wolf, S. & Gerwert, K. (2011) Proton transfer via a transient linear water-molecule chain in a membrane protein. Proc. Natl. Acad. Sci. USA 108, 11435–11439.
Gelis, L., Wolf, S., Hatt, H., Neuhaus, E. M. & Gerwert, K. (2012) Prediction of a ligand-binding niche within a human olfactory receptor by combining site-directed mutagenesis with dynamic homology modeling. Angew. Chem. Int. Ed. Engl. 51, 1274–1278.
Kuhne, J. et al. Early formation of the ion-conducting pore in channelrhodopsin-2. (2015). Angew. Chem. Int. Ed. Engl. 54, 4953–4957.