Diversity of Chlamydomonas Channelrhodopsins

Abstract

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 red-shifted 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 red-shifted spectra and the lack of fast inactivation in CaChR1- and CyChR1-generated currents are features desirable for optogenetics applications.

Synthetic retinal analogues modify the spectral and kinetic characteristics of microbial rhodopsin optogenetic tools

“…A recent focus of opsin research has been to shift its action spectrum, either to long-wavelength absorption 19,[29][30][31][32][33] or to wavelengths below its maximum of 470 nm (refs 33,34). This will facilitate the independent activation of different cell types or allow to combine photoactivation with the imaging of neuronal activity using genetically encoded Ca 2 þ indicators (GECIs) [35][36][37][38] .…”

Changes in the hydrogen-bonding strength of internal water molecules and cysteine residues in the conductive state of channelrhodopsin-1

“…47 Among them, ChR1 from Chlamydomonas augustae, CaChR1, shows some favorable properties to compete with CrChR2 as a tool to depolarize the membrane potential of a host cell by light: a redshifted absorption maximum (see Fig. S1 of the supplementary material 59 ) and a lower inactivation level under sustained illumination.…”

Platymonas subcordiformis Channelrhodopsin-2 (PsChR2) Function

“…However, the extent of peak current inactivation or desensitization is less and the recovery to repetitive illumination steps is faster than those of CrChR2 (10). The time course of the current induced by a laser flash lacks the ultrafast transient signals seen in rhodopsin proton pumps and CrChR1 associated with proton transfer from the retinylidene Schiff base (18,19) and follows the time dependence of the absorption transient assigned to an unidentified red-shifted intermediate (10). These properties of PsChR2, mentioned above briefly, make this protein a desirable subject for studies aimed at connecting the channel current kinetics to light-induced intermediates.…”