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, Ca2+-selective ChRs have not been reported to date. Here, we analyse ChRs and mutant derivatives with regard to their Ca2+ permeability and improve their Ca2+ 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 Ca2+ permeation at negative voltage and low extracellular Ca2+ concentrations. In cultured cells and neurons, CapChR2 reliably increases intracellular Ca2+ concentrations. Moreover, CapChR2 can robustly trigger Ca2+ signalling in hippocampal neurons. When expressed together with genetically encoded Ca2+ indicators in Drosophila melanogaster mushroom body output neurons, CapChRs mediate light-evoked Ca2+ entry in brain explants.

Gating and ion selectivity of Channelrhodopsins are critical for photo-activated orientation of Chlamydomonas as shown by in vivo point mutation.

The green unicellular alga Chlamydomonas reinhardtii with two photoreceptors called channelrhodopsins is a model organism that gave birth to a new scientific field of biomedical studies, optogenetics. Although channelrhodopsins are helping to decipher the activity of the human brain, their functionality has never been extensively studied in the organism of origin, mainly due to the difficulties connected to reverse genetic interventions. In this study, we present a CRISPR-Cas9-based technique that enables a precise in vivo exchange of single amino acids in a selected gene. To shed light on the function of channelrhodopsins ChR1 (C1) and ChR2 (C2) in vivo, we deleted both channelrhodopsins independently in the wild-type strain and introduced point mutations in the remaining channel, causing modified photocycle kinetics and ion selectivity. The mutated strains, ΔC1C2-E123T, ΔC1C2-E90R and ΔC1C2-E90Q, showed about 100-fold decrease in photosensitivity, a reduced photophobic response and faster light adaptation rates due to accelerated photocycle kinetics and reduced Ca2+ conductance. Moreover, the ΔC1C2-E90Q with an additionally reduced H+ permeability produced an electrical response only in the presence of Na+ ions, highlighting a contribution and importance of H+ conductance to photocurrents in the wild-type algae. Finally, in the ΔC1C2-E90R strain with the channelrhodopsin selectivity converted to anions, no photo-responses were detected. We conclude that the precise photocycle kinetics and the particular ion selectivity of channelrhodopsins are the key parameters for efficient phototaxis in low light conditions.

QuasAr Odyssey: the origin of fluorescence and its voltage sensitivity in microbial rhodopsins.

Rhodopsins had long been considered non-fluorescent until a peculiar voltage-sensitive fluorescence was reported for archaerhodopsin-3 (Arch3) derivatives. These proteins named QuasArs have been used for imaging membrane voltage changes in cell cultures and small animals, but they could not be applied in living rodents. To develop the next generation of sensors, it is indispensable to first understand the molecular basis of the fluorescence and its modulation by the membrane voltage. Based on spectroscopic studies of fluorescent Arch3 derivatives, we propose a unique photo-reaction scheme with extended excited-state lifetimes and inefficient photoisomerization. Molecular dynamics simulations of Arch3, of the Arch3 fluorescent derivative Archon1, and of several its mutants have revealed different voltage-dependent changes of the hydrogen-bonding networks including the protonated retinal Schiff-base and adjacent residues. Experimental observations suggest that under negative voltage, these changes modulate retinal Schiff base deprotonation and promote a decrease in the populations of fluorescent species. Finally, we identified molecular constraints that further improve fluorescence quantum yield and voltage sensitivity.