Blue- versus green-absorbing anion channelrhodopsins, essential tools in optogenetics, differ fundamentally in gating...

The genomes of many protists encode at least one pair of spectrally distinct cation or anion channelrhodopsins. Two cation channelrhodopsins (CCRs) initiate different transduction cascades in Chlamydomonas. Two Guillardia anion channelrhodopsins (ACRs) are emerging molecular tools for optical inhibition of neuronal firing, but their functions in the source organism remain unclear. Furthermore, GtACR2 remains poorly investigated compared to GtACR1, although its faster channel kinetics makes it preferable for optogenetic control of neurons firing at high frequencies. Using patch-clamp recording in mammalian cells, photochemical characterization of purified proteins, and mutational analysis, we found a fundamental mechanistic difference between blue-absorbing GtACR2 and green-absorbing GtACR1, which may indicate different functions in the alga. GtACR2 exhibits only one channel gating mechanism, unlike GtACR1, in which we had detected two mechanisms. GtACR2's faster channel closing strongly depends on the holding voltage, and its photocycle shows considerable intermediate reversibility. Photocurrents evoked by continuous light pulses reflecting secondary photochemistry reveal further differences between GtACR1 and GtACR2. Mutagenetic replacement of five divergent GtACR2 residues with those of GtACR1 reproduced the latter's spectral sensitivity and slow biphasic channel closing. Only one GtACR2 mutation, R129C, which individually caused the most substantial red shift, restored the gating mechanism typical of GtACR1. GtACR2, but not GtACR1, exhibited a change in the F^-/Cl^- relative permeability during the single-turnover photocycle. Comparison to Chlamydomonas CCRs and ACRs from other protists suggests that these differences between blue- and green-absorbing ChRs are a general rule, which can guide further development of optogenetic tools.