Calcium-permeable channelrhodopsins for the photocontrol of calcium signalling
Rodrigo G. Fernandez Lahore, Niccolò Paolo Pampaloni, Enrico Schiewer, M.‐Marcel Heim, Linda Tillert, Johannes Vierock, Johannes Oppermann, Jakob Walther, Dietmar Schmitz, David Owald, Andrew J.R. Plested, Benjamin R. Rost, Peter Hegemann
Abstract
Abstract 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.