TY  - JOUR
A1  - Scheib, Ulrike
A1  - Broser, Matthias
A1  - Constantin, Oana M.
A1  - Yang, Shang
A1  - Gao, Shiqiang
A1  - Mukherjee, Shatanik
A1  - Stehfest, Katja
A1  - Nagel, Georg
A1  - Gee, Christine E.
A1  - Hegemann, Peter
T1  - Rhodopsin-cyclases for photocontrol of cGMP/cAMP and 2.3 Å structure of the adenylyl cyclase domain
JF  - Nature Communications
N2  - The cyclic nucleotides cAMP and cGMP are important second messengers that orchestrate fundamental cellular responses. Here, we present the characterization of the rhodopsinguanylyl cyclase from Catenaria anguillulae (CaRhGC), which produces cGMP in response to green light with a light to dark activity ratio > 1000. After light excitation the putative signaling state forms with tau = 31 ms and decays with tau = 570 ms. Mutations (up to 6) within the nucleotide binding site generate rhodopsin-adenylyl cyclases (CaRhACs) of which the double mutated YFP-CaRhAC (E497K/C566D) is the most suitable for rapid cAMP production in neurons. Furthermore, the crystal structure of the ligand-bound AC domain (2.25 angstrom) reveals detailed information about the nucleotide binding mode within this recently discovered class of enzyme rhodopsin. Both YFP-CaRhGC and YFP-CaRhAC are favorable optogenetic tools for non-invasive, cell-selective, and spatio-temporally precise modulation of cAMP/cGMP with light.
KW  - Enzymes
KW  - Molecular biophysics
KW  - Molecular neuroscience
KW  - X-ray crystallography
Y1  - 2018
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-228517
VL  - 9
ER  - 
TY  - JOUR
A1  - Beck, Sebastian
A1  - Yu-Strzelczyk, Jing
A1  - Pauls, Dennis
A1  - Constantin, Oana M.
A1  - Gee, Christine E.
A1  - Ehmann, Nadine
A1  - Kittel, Robert J.
A1  - Nagel, Georg
A1  - Gao, Shiqiang
T1  - Synthetic light-activated ion channels for optogenetic activation and inhibition
JF  - Frontiers in Neuroscience
N2  - Optogenetic manipulation of cells or living organisms became widely used in neuroscience following the introduction of the light-gated ion channel channelrhodopsin-2 (ChR2). ChR2 is a non-selective cation channel, ideally suited to depolarize and evoke action potentials in neurons. However, its calcium (Ca2\(^{2+}\)) permeability and single channel conductance are low and for some applications longer-lasting increases in intracellular Ca\(^{2+}\) might be desirable. Moreover, there is need for an efficient light-gated potassium (K\(^{+}\)) channel that can rapidly inhibit spiking in targeted neurons. Considering the importance of Ca\(^{2+}\) and K\(^{+}\) in cell physiology, light-activated Ca\(^{2+}\)-permeant and K\(^{+}\)-specific channels would be welcome additions to the optogenetic toolbox. Here we describe the engineering of novel light-gated Ca\(^{2+}\)-permeant and K\(^{+}\)-specific channels by fusing a bacterial photoactivated adenylyl cyclase to cyclic nucleotide-gated channels with high permeability for Ca\(^{2+}\) or for K\(^{+}\), respectively. Optimized fusion constructs showed strong light-gated conductance in Xenopus laevis oocytes and in rat hippocampal neurons. These constructs could also be used to control the motility of Drosophila melanogaster larvae, when expressed in motoneurons. Illumination led to body contraction when motoneurons expressed the light-sensitive Ca\(^{2+}\)-permeant channel, and to body extension when expressing the light-sensitive K\(^{+}\) channel, both effectively and reversibly paralyzing the larvae. Further optimization of these constructs will be required for application in adult flies since both constructs led to eclosion failure when expressed in motoneurons.
KW  - optogenetics
KW  - calcium
KW  - potassium
KW  - bPAC
KW  - CNG channel
KW  - cAMP
KW  - Drosophila melanogaster motoneuron
KW  - rat hippocampal neurons
Y1  - 2018
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-177520
VL  - 12
IS  - 643
ER  -