A second photoactivatable state of the anion-conducting channelrhodopsin G\(\it t\)ACR1 empowers persistent activity

Optogenetics is a method to regulate cells, tissues and organisms using light. It is applied to study neurons and to develop diagnostic and therapeutic tools for neuron-related diseases. The cation-conducting channelrhodopsin ChR2 triggers photoinduced depolarization of neuronal cells but generates lower ion currents due to the \(\it syn\)-pathway of its branched photocycle. In contrast, the homologous anion-conducting ACR1 from \(\textit {Guillardia theta}\) (G\(\it t\)ACR1), exhibits high photocurrents. Here, we investigate the mechanistic cause for the observed high photocurrents in G\(\it t\)ACR1 using FTIR spectroscopy. Unexpectedly, we discovered that the \(\it O\) intermediate of G\(\it t\)ACR1 is photoactivable, allowing for fast and efficient channel reopening. Our vibrational spectra show a photocyclic reaction sequence after \(\it O\) excitation similar to the ground state photocycle but with slightly altered channel conformation and protonation states. Our results provide deeper insights into the gating mechanism of channelrhodopsins and pave the way to advance the development of optimized optogenetic tools in future.