Jonas Wietek, Silvia Rodriguez-Rozada, Janine Tutas, Federico Tenedini,
Christiane Grimm, Thomas G. Oertner, Peter Soba, Peter Hegemann &
J. Simon Wiegert
a) Institute for Biology, Experimental Biophysics, Humboldt-Universität zu Berlin, 11 Invalidenstraße 42, 10115 Berlin, Germany.12 b) Research Group Neuronal Patterning and Connectivity, Center for Molecular Neurobiology
13 Hamburg, Falkenried 94, 20251 Hamburg, Germany.14 c) Institute for Synaptic Physiology, Center for Molecular Neurobiology Hamburg, Falkenried15 94, 20251 Hamburg, Germany.16 d) Research Group Synaptic Wiring and Information Processing, Center for Molecular 17 Neurobiology Hamburg, Falkenried 94, 20251 Hamburg, Germany18 1) Correspondence to: email@example.com, phone: +49 40 7410 55354
Genetic engineering of natural light-gated ion channels has proven a powerful way to generate optogenetic tools for a wide variety of applications. In recent years, blue-light activated engineered anion-conducting channelrhodopsins (eACRs) have been developed, improved, and were successfully applied in vivo. We asked whether the approaches used to create eACRs can be transferred to other well-characterized cation-conducting channelrhodopsins (CCRs) to obtain eACRs with a broad spectrum of biophysical properties. We generated 22 variants using two conversion strategies applied to 11 CCRs and screened them for membrane expression, photocurrents and anion selectivity. We obtained two novel eACRs, Phobos and Aurora, with blue- and red-shifted action spectra and photocurrents similar to existing eACRs. Furthermore, step-function mutations greatly enhanced the cellular operational light sensitivity due to a slowed-down photocycle. These bi-stable eACRs can be reversibly toggled between open and closed states with brief light pulses of different wavelengths. All new eACRs reliably inhibited action potential firing in pyramidal CA1 neurons. In Drosophila larvae, eACRs conveyed robust and specific light-dependent inhibition of locomotion and nociception.
“During free locomotion, eACRs were activated by illumination with 525 nm light from a RGB-BL-S-Q-1 R LED backlight (Phlox, Aix-en-Provence, France) for Aurora, or 460, 470 and 595 nm light from a pE-4000 (CoolLED) coupled to a light guide with custom collimator lenses for Phobos and PhobosCA.”
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