TY  - JOUR
A1  - Tian, Yuehui
A1  - Yang, Shang
A1  - Nagel, Georg
A1  - Gao, Shiqiang
T1  - Characterization and modification of light-sensitive phosphodiesterases from choanoflagellates
JF  - Biomolecules
N2  - Enzyme rhodopsins, including cyclase opsins (Cyclops) and rhodopsin phosphodiesterases (RhoPDEs), were recently discovered in fungi, algae and protists. In contrast to the well-developed light-gated guanylyl/adenylyl cyclases as optogenetic tools, ideal light-regulated phosphodiesterases are still in demand. Here, we investigated and engineered the RhoPDEs from Salpingoeca rosetta, Choanoeca flexa and three other protists. All the RhoPDEs (fused with a cytosolic N-terminal YFP tag) can be expressed in Xenopus oocytes, except the AsRhoPDE that lacks the retinal-binding lysine residue in the last (8th) transmembrane helix. An N296K mutation of YFP::AsRhoPDE enabled its expression in oocytes, but this mutant still has no cGMP hydrolysis activity. Among the RhoPDEs tested, SrRhoPDE, CfRhoPDE1, 4 and MrRhoPDE exhibited light-enhanced cGMP hydrolysis activity. Engineering SrRhoPDE, we obtained two single point mutants, L623F and E657Q, in the C-terminal catalytic domain, which showed ~40 times decreased cGMP hydrolysis activity without affecting the light activation ratio. The molecular characterization and modification will aid in developing ideal light-regulated phosphodiesterase tools in the future.
KW  - choanoflagellates
KW  - optogenetics
KW  - rhodopsin phosphodiesterase (RhoPDE)
KW  - cGMP
Y1  - 2022
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-254769
SN  - 2218-273X
VL  - 12
IS  - 1
ER  - 
TY  - JOUR
A1  - vom Dahl, Christian
A1  - Müller, Christoph Emanuel
A1  - Berisha, Xhevat
A1  - Nagel, Georg
A1  - Zimmer, Thomas
T1  - Coupling the cardiac voltage-gated sodium channel to channelrhodopsin-2 generates novel optical switches for action potential studies
JF  - Membranes
N2  - Voltage-gated sodium (Na\(^+\)) channels respond to short membrane depolarization with conformational changes leading to pore opening, Na\(^+\) influx, and action potential (AP) upstroke. In the present study, we coupled channelrhodopsin-2 (ChR2), the key ion channel in optogenetics, directly to the cardiac voltage-gated Na\(^+\) channel (Na\(_v\)1.5). Fusion constructs were expressed in Xenopus laevis oocytes, and electrophysiological recordings were performed by the two-microelectrode technique. Heteromeric channels retained both typical Na\(_v\)1.5 kinetics and light-sensitive ChR2 properties. Switching to the current-clamp mode and applying short blue-light pulses resulted either in subthreshold depolarization or in a rapid change of membrane polarity typically seen in APs of excitable cells. To study the effect of individual K\(^+\) channels on the AP shape, we co-expressed either K\(_v\)1.2 or hERG with one of the Na\(_v\)1.5-ChR2 fusions. As expected, both delayed rectifier K\(^+\) channels shortened AP duration significantly. K\(_v\)1.2 currents remarkably accelerated initial repolarization, whereas hERG channel activity efficiently restored the resting membrane potential. Finally, we investigated the effect of the LQT3 deletion mutant ΔKPQ on the AP shape and noticed an extremely prolonged AP duration that was directly correlated to the size of the non-inactivating Na\(^+\) current fraction. In conclusion, coupling of ChR2 to a voltage-gated Na\(^+\) channel generates optical switches that are useful for studying the effect of individual ion channels on the AP shape. Moreover, our novel optogenetic approach provides the potential for an application in pharmacology and optogenetic tissue-engineering.
KW  - optogenetics
KW  - channelrhodopsin
KW  - voltage-gated Na\(^+\) channel
KW  - action potential
KW  - delayed rectifier potassium channel
KW  - hERG
KW  - long QT syndrome
Y1  - 2022
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-288228
SN  - 2077-0375
VL  - 12
IS  - 10
ER  -