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.
Highlights
Channelrhodopsins are widely used in optogenetic applications
We have demonstrated that upon light activation, the dark-adapted ground state CrChR2 either undergoes a photocycle with an efficiently conducting open state (Fig. 1a, left) or enters another light-adapted ground state, which leads to an alternative photocycle characterized by a less efficient ion conduction (Fig. 1a, right)
The photocycle of GtACR1 was studied by focusing on the early transitions related to gate opening by UV/VIS and vibrational spectroscopy
Summary
Channelrhodopsins are widely used in optogenetic applications. High photocurrents and low current inactivation levels are desirable. CCRs produce membrane depolarization (generation of action potentials) to activate cells, whereas ACRs hyperpolarize membranes (suppression of action potentials) to deactivate cells[19] To perform these tasks, ideal optogenetic channels would comprise features such as (i) high sensitivity to activating light, (ii) fast channel gating (i.e., fast opening and closing), and (iii) intense photocurrents. The photocurrent of CrChR2 exhibits an initial sharp peak that rapidly decreases to a much lower but stable level[23] (Supplementary Fig 1) The syncycle comprises a poorly conducting open state that slowly decays back to either the dark-adapted or light-adapted ground state and accumulates under continuous illumination, causing a substantial decrease in the photocurrent of CrChR2 (Supplementary Fig. 1). The decay of L to M makes GtACR1 non-conducting
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