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  - 
TY  - JOUR
A1  - Duan, Xiaodong
A1  - Nagel, Georg
A1  - Gao, Shiqiang
T1  - Mutated channelrhodopsins with increased sodium and calcium permeability
JF  - Applied Sciences
N2  - (1) Background: After the discovery and application of Chlamydomonas reinhardtii channelrhodopsins, the optogenetic toolbox has been greatly expanded with engineered and newly discovered natural channelrhodopsins. However, channelrhodopsins of higher Ca\(^{2+}\) conductance or more specific ion permeability are in demand. (2) Methods: In this study, we mutated the conserved aspartate of the transmembrane helix 4 (TM4) within Chronos and PsChR and compared them with published ChR2 aspartate mutants. (3) Results: We found that the ChR2 D156H mutant (XXM) showed enhanced Na\(^+\) and Ca\(^{2+}\) conductance, which was not noticed before, while the D156C mutation (XXL) influenced the Na\(^+\) and Ca\(^{2+}\) conductance only slightly. The aspartate to histidine and cysteine mutations of Chronos and PsChR also influenced their photocurrent, ion permeability, kinetics, and light sensitivity. Most interestingly, PsChR D139H showed a much-improved photocurrent, compared to wild type, and even higher Na+ selectivity to H\(^+\) than XXM. PsChR D139H also showed a strongly enhanced Ca\(^{2+}\) conductance, more than two-fold that of the CatCh. (4) Conclusions: We found that mutating the aspartate of the TM4 influences the ion selectivity of channelrhodopsins. With the large photocurrent and enhanced Na\(^+\) selectivity and Ca\(^{2+}\) conductance, XXM and PsChR D139H are promising powerful optogenetic tools, especially for Ca\(^{2+}\) manipulation.
KW  - optogenetics
KW  - channelrhodopsins
KW  - sodium
KW  - calcium
KW  - DC gate
Y1  - 2019
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-197435
SN  - 2076-3417
VL  - 9
IS  - 4
ER  -