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 calcium-permeable channelrhodopsins, 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.