Time-Resolved Analysis of Protonation Dynamics in Channelrhodopsin-2

by Tom Resler

Institution: Freie Universitt Berlin
Year: 2017
Posted: 02/01/2018
Record ID: 2154066
Full text PDF: http://edocs.fu-berlin.de/diss/receive/FUDISS_thesis_000000104959


As presented in my thesis, we made enormous progress in resolving the molecular details of the photoreaction of the light-gated ion channel channelrhodopsin-2 (ChR2), by applying time-resolved molecular spectroscopy. I have shown that D253 is the primary proton acceptor and D156 the internal proton donor of the retinal Schiff base of ChR2. The reprotonation of the latter residue constitutes the molecular determinant for channel closure. Upon H/D exchange of the solvent, the kinetic and vibrational isotope effects of these reactions and that of E90, a key residue in ion selectivity, were determined. My experiments with a pH indicating dye show that the de- and reprotonation of E90 takes place during the life time of the desensitized P4 state. Thus, other conclusions that E90 plays a key role in channel opening are clearly ruled out. Light-adapted ChR2 contains a mixture of all-trans and 13-cis retinal. I contributed to time-resolved analysis of the E123T variant and we could show that the 13-cis photocycle does not lead to channel opening. Furthermore, the channel opening tallies with transient changes in the amide I vibration, identified as water influx to hydrate the peptide backbone. These findings from ChR2 were confirmed by ex- pediments on variants of ChR2 that exhibit accelerated (E123T) or delayed (D156E) channel opening. The associated activation volumes are monitored and quantified by flash photolysis under hydrostatic pressures. Finally, the loss-of-function variant R120H was investigated, which exhibits wild-type photocycle kinetics but channel activity is blocked. Time-resolved IR spectroscopy covering almost 13 decades in time are presented (from 500 fs to s). An assignment of the arginine band was not feasible, but the possible role of His-Arg interactions were discussed and I demonstrated that R120 is not part of the proton release complex. Although the mechanistic link between proton transfer reactions and channel func- tion remain elusive, I could show that protonation dynamics are crucial in the ChR2 photocylce and are related to ion conductance. ChR2 is the mayor tool in optoge- netics and a detailed understanding of this ion channel is supportive for medical application. Furthermore, it is a good model system for other ion channels, because in ChR2 ion permeation is triggered repeatable, non-invasive and reproducible by light, which allows the observation of channel on- and off-gating with high temporal and spacial resolution. In meiner Dissertation prsentiere ich unsere Fortschritte bei der molekularen Erforschung des lichtgesteuerten Innenkanals Kanalrhodopsin-2 (ChR2) mittels zeitaufgelster Spektroskopie. Ich konnte zeigen, dass D253 der primre Protonenakzeptor und D156 der primre Protonendonor der Schiffschen Base in ChR2 ist. Die Reprotonierung von D156 geht einher mit der Kanalschlieung. Durch Messungen in H2O und D2O konnten der kinetische und spektrale Isotopeneffekt dieser Reaktionen und von E90 bestimmt werden. E90 is wichtig fr die Ionenselektivitt, aber eine De- und