Tuning wavelength and G protein specificity of Melanopsin for optogenetic control of G protein signaling pathways.
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).
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.
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.