Time-resolved spectroscopic and electrophysiological data reveal insights in the gating mechanism of anion channelrhodopsin

Abstract

Channelrhodopsins are widely used in optogenetic applications. High photocurrents and low current inactivation levels are desirable. Two parallel photocycles evoked by different retinal conformations cause cation-conducting channelrhodopsin-2 (CrChR2) inactivation: one with efficient conductivity; one with low conductivity. Given the longer half-life of the low conducting photocycle intermediates, which accumulate under continuous illumination, resulting in a largely reduced photocurrent. Here, we demonstrate that for channelrhodopsin-1 of the cryptophyte Guillardia theta (GtACR1), the highly conducting C = N-anti-photocycle was the sole operating cycle using time-resolved step-scan FTIR spectroscopy. The correlation between our spectroscopic measurements and previously reported electrophysiological data provides insights into molecular gating mechanisms and their role in the characteristic high photocurrents. The mechanistic importance of the central constriction site amino acid Glu-68 is also shown. We propose that canceling out the poorly conducting photocycle avoids the inactivation observed in CrChR2, and anticipate that this discovery will advance the development of optimized optogenetic tools.

Proton transfer pathway in anion channelrhodopsin-1

“…The calculated protonation pattern shows that Asp234 is deprotonated in the wild-type Gt ACR1 even using the MD-generated conformations with protonated Glu68/protonated Asp234 or deprotonated Glu68/protonated Asp234 ( Table 1 , Supplementary file 1B , Figure 2—figure supplement 1 ). These results suggest that deprotonation of Asp234, ‘the only residue directly interacting with the protonated Schiff base ( Li et al, 2019 )’, is a prerequisite to stabilize the protonated Schiff base, as suggested by Dreier et al, 2021 .…”

“…The C=O stretching frequencies of 1740 (–)/1732 (+) cm –1 for protonated Asp234 at 77 K observed by Kim et al, 2018 were not observed at 293 K by Dreier et al, 2021 . In addition, MD simulations indicated that the H-bond network that involves Tyr72, Tyr207, and Asp234 was stable with deprotonated Asp234 but unstable with protonated Asp234 ( Dreier et al, 2021 ). Indeed, the presence of deprotonated Asp234 was already suggested based on the homology modeling of Gt ACR2 ( Kojima et al, 2018 ) before the crystal structures of Gt ACR1 were reported.…”

“…Indeed, the C=C stretching frequency of the retinal is not significantly affected upon the E68Q and D234N mutations in resonance Raman spectroscopy, which implies that the electrostatic interaction between the retinal and protein environment remains unchanged ( Yi et al, 2016 ). In addition, the C=O stretching frequency for a protonated carboxylate, which is observed in the wild-type Gt ACR1, disappears in the E68Q ( Yi et al, 2017 ; Dreier et al, 2021 ) and D234N ( Kim et al, 2018 ) Gt ACR1s according to Fourier transform infrared (FTIR) spectroscopy analysis.…”

“…Recently, Dreier et al proposed that Asp234 is deprotonated in the dark and acts as a counterion according to FTIR measurements and molecular dynamics (MD) simulations ( Dreier et al, 2021 ). The C=O stretching frequencies of 1740 (–)/1732 (+) cm –1 for protonated Asp234 at 77 K observed by Kim et al, 2018 were not observed at 293 K by Dreier et al, 2021 .…”

“…Recently, Dreier et al proposed that Asp234 is deprotonated in the dark and acts as a counterion according to FTIR measurements and molecular dynamics (MD) simulations ( Dreier et al, 2021 ). The C=O stretching frequencies of 1740 (–)/1732 (+) cm –1 for protonated Asp234 at 77 K observed by Kim et al, 2018 were not observed at 293 K by Dreier et al, 2021 . In addition, MD simulations indicated that the H-bond network that involves Tyr72, Tyr207, and Asp234 was stable with deprotonated Asp234 but unstable with protonated Asp234 ( Dreier et al, 2021 ).…”