Unifying photocycle model for light adaptation and temporal evolution of cation conductance in channelrhodopsin-2

Abstract

Although channelrhodopsin (ChR) is a widely applied light-activated ion channel, important properties such as light adaptation, photocurrent inactivation, and alteration of the ion selectivity during continuous illumination are not well understood from a molecular perspective. Herein, we address these open questions using single-turnover electrophysiology, time-resolved step-scan FTIR, and Raman spectroscopy of fully dark-adapted ChR2. This yields a unifying parallel photocycle model integrating now all so far controversial discussed data. In dark-adapted ChR2, the protonated retinal Schiff base chromophore (RSBH+) adopts an all-trans,C=N-anti conformation only. Upon light activation, a branching reaction into either a 13-cis,C=N-anti or a 13-cis,C=N-syn retinal conformation occurs. The anti-cycle features sequential H+ and Na+ conductance in a late M-like state and an N-like open-channel state. In contrast, the 13-cis,C=N-syn isomer represents a second closed-channel state identical to the long-lived P480 state, which has been previously assigned to a late intermediate in a single-photocycle model. Light excitation of P480 induces a parallel syn-photocycle with an open-channel state of small conductance and high proton selectivity. E90 becomes deprotonated in P480 and stays deprotonated in the C=N-syn cycle. Deprotonation of E90 and successive pore hydration are crucial for late proton conductance following light adaptation. Parallel anti- and syn-photocycles now explain inactivation and ion selectivity changes of ChR2 during continuous illumination, fostering the future rational design of optogenetic tools.

MerMAIDs: a family of metagenomically discovered marine anion-conducting and intensely desensitizing channelrhodopsins

“…Hence, stationary photocurrents might be potentially a cause of a branched photocycle instead, in which a second photoactive closed state can be populated from the initial dark state by photon absorption, as discussed for various CCRs 7,8,[51][52][53] . As recently substantiated experimentally for CrChR2, the retinal in both parallel photocycles differ, adopting either a syn or an anti conformation 10 . Conceivably, C84 could suppress a C=N anti to syn isomerization in MerMAID1 and therefore prevent population of parallel syn-photocycle that could account for a second conducting state and stationary photocurrents.…”

“…For light titration experiments, photocurrents were induced for 2 s at −60 mV, and light was attenuated using ND filters (SCHOTT, Mainz, Germany) inserted into the light path using a motorized, software-controlled filter wheel (FW212C, Thorlabs, Newton, NJ). Single-turnover experiments were performed with the above mentioned setup described elsewhere 10,76 . An Opolette HE 355 LD Nd:YAG laser/OPO system (OPOTEK, Carlsbad,CA) served as pulsed laser light source.…”

“…The degree and kinetics of desensitization differ among ChRs and depend on pH, membrane voltage as well as light intensity and color, with typically ≤70% amplitude reduction 1,2,7 . Photocurrent decrease via desensitization has been explained by accumulation of late non-conducting photocycle intermediates and by an alternative photocycle exhibiting low cation conductance [7][8][9][10] .…”

Viral channelrhodopsins: calcium-dependent Na+/K+ selective light-gated channels

“…In addition, the VR1 group has a signature topological feature, namely, an extended TM4 helix that consist of it transmembrane (TM4) and membrane-associated parts (ICL2) and has not been previously observed in characterized microbial rhodopsins (Figure 1b). Despite the overall low structural similarity with cryptophyte cation-conducting channelrhodopsins (Figure 1d), viral rhodopsins from group 1 retain the two highly conservative glutamates in TM2 (E44 and E51 in OLPVR1,and E83 and E90 in CrChR2) that have been shown to be critical for CrChR2 ion channelling (Kuhne et al, 2019;Wietek et al, 2014).…”

“…Unlike other channelrhodopsins, OLPVR1 lacks a detectable M-state, that is generally associated with the ion-conducting state of the protein (Kuhne et al, 2019). For viral channelrhodopsins, the equilibrium between L-like and N-like states is the major candidate for the ion-conducting state, where the structural changes in the protein may occur.…”