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

Abstract

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 <i>Chlamydomonas</i>. Two <i>Guillardia</i> anion channelrhodopsins (ACRs) are emerging molecular tools for optical inhibition of neuronal firing, but their functions in the source organism remain unclear. Furthermore, <i>Gt</i>ACR2 remains poorly investigated compared to <i>Gt</i>ACR1, 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 <i>Gt</i>ACR2 and green-absorbing <i>Gt</i>ACR1, which may indicate different functions in the alga. <i>Gt</i>ACR2 exhibits only one channel gating mechanism, unlike <i>Gt</i>ACR1, in which we had detected two mechanisms. <i>Gt</i>ACR2'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 <i>Gt</i>ACR1 and <i>Gt</i>ACR2. Mutagenetic replacement of five divergent <i>Gt</i>ACR2 residues with those of <i>Gt</i>ACR1 reproduced the latter's spectral sensitivity and slow biphasic channel closing. Only one <i>Gt</i>ACR2 mutation, R129C, which individually caused the most substantial red shift, restored the gating mechanism typical of <i>Gt</i>ACR1. <i>Gt</i>ACR2, but not <i>Gt</i>ACR1, exhibited a change in the F^-/Cl^- relative permeability during the single-turnover photocycle. Comparison to <i>Chlamydomonas</i> 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.