Calcium-permeable channelrhodopsins for the photocontrol of calcium signalling

Channelrhodopsins are light-gated ion channels used to control excitability of designated cells in large networks with high spatiotemporal resolution. While ChRs selective for H + , Na + , K + and anions have been discovered or engineered, Ca 2+ -selective ChRs have not been reported to date. Here, we analyse ChRs and mutant derivatives with regard to their Ca 2+ permeability and improve their Ca 2+ affinity by targeted mutagenesis at the central selectivity filter. The engineered channels, termed CapChR1 and CapChR2 for ca lcium- p ermeable ch annel r hodopsins, exhibit reduced sodium and proton conductance in connection with strongly improved Ca 2+ permeation at negative voltage and low extracellular Ca 2+ concentrations. In cultured cells and neurons, CapChR2 reliably increases intracellular Ca 2+ concentrations. Moreover, CapChR2 can robustly trigger Ca 2+ signalling in hippocampal neurons. When expressed together with genetically encoded Ca 2+ indicators in Drosophila melanogaster mushroom body output neurons, CapChRs mediate light-evoked Ca 2+ entry in brain explants.