Artificial Anion Conducting Channelrhodopsins with Tuned Spectra, Modified Kinetics and Enhanced Light Sensitivity

Prior to the discovery of natural anion conducting channelrhodopsins (nACRs), artificial anion conducting channelrhodopsins (aACRs) have been developed, improved and were successfully applied in vivo. We asked whether the approaches used to create aACRs can be transferred to other well-characterized cation-conducting channelrhodopsins to obtain aACRs with a broader variation of biophysical properties. The screen of 18 different constructs yielded two new aACRs, termed Phobos and Aurora, which express well in neurons and provide sufficient photocurrents for efficient silencing. They can be activated with far blue or far red light, respectively, making them suitable for combination with other spectrally distinct actuators or sensors of the optogenetic toolbox. Furthermore, introduction of step function mutations slowed down the photocycle of the new aACRs up to 10000-fold, thus greatly enhancing light sensitivity. These light-switchable versions of Phobos and Aurora can further be reversibly toggled between open and closed states with brief light pulses of different wavelengths. Both variants and their step function mutants showed perfect selectivity for chloride ions and where tested in hippocampal slice cultures, reliably inhibiting action potential firing. In drosophila larvae, the new aACR variants showed robust and specific light-dependent in vivo inhibition of locomotion and nociception. Ongoing experiments will reveal in vivo aACR functionality in rodents and adult drosophila.