Diversity of Chlamydomonas Channelrhodopsins

Channelrhodopsins act as photoreceptors for control of motility behavior in flagellates and are widely used as genetically targeted tools to optically manipulate the membrane potential of specific cell populations (“optogenetics”). The first two channelrhodopsins were obtained from the model organism Chlamydomonas reinhardtii (CrChR1 and CrChR2). By homology cloning we identified three new channelrhodopsin sequences from the same genus, CaChR1, CyChR1 and CraChR2, from C. augustae, C. yellowstonensis and C. raudensis, respectively. CaChR1 and CyChR1 were functionally expressed in HEK293 cells, where they acted as light‐gated ion channels similar to CrChR1. However, both, which are similar to each other, differed from CrChR1 in current kinetics, inactivation, light intensity dependence, spectral sensitivity and dependence on the external pH. These results show that extensive channelrhodopsin diversity exists even within the same genus, Chlamydomonas. The maximal spectral sensitivity of CaChR1 was at 520 nm at pH 7.4, about 40 nm redshifted as compared to that of CrChR1 under the same conditions. CaChR1 was successfully expressed in Pichia pastoris and exhibited an absorption spectrum identical to the action spectrum of CaChR1‐generated photocurrents. The redshifted spectra and the lack of fast inactivation in CaChR1‐ and CyChR1‐generated currents are features desirable for optogenetics applications. We report cloning of three new channelrhodopsin homologs from three Chlamydomonas species and their characterization upon heterologous expression in HEK293 cells and Pichia pastoris. Channelrhodopsins from Chlamydomonas augustae and C. yellowstonensis belong to the same class as the earlier identified ChR1 from C. reinhardtii, but their channel activity differs from that of ChR1 in kinetics, inactivation, light intensity dependence, spectral sensitivity and pH dependence, which reveals extensive channelrhodopsin diversity within the same genus. The redshifted spectra and the lack of fast inactivation in currents generated by the new channelrhodopsins are features desirable for optogenetics applications.