Abstract
We previously reported a cation channelrhodopsin, Gt_CCR4, which is one of the 44 types of microbial rhodopsins from a cryptophyte flagellate, Guillardia theta. Due to the modest homology of amino acid sequences with a chlorophyte channelrhodopsin such as Cr_ChR2 from Chlamydomonas reinhardtii, it has been proposed that a family of cryptophyte channelrhodopsin, including Gt_CCR4, has a distinct molecular mechanism for channel gating and ion permeation. In this study, we compared the photocurrent properties, cation selectivity and kinetics between well-known Cr_ChR2 and Gt_CCR4 by a conventional path clamp method. Large and stable light-induced cation conduction by Gt_CCR4 at the maximum absorbing wavelength (530 nm) was observed with only small inactivation (15%), whereas the photocurrent of Cr_ChR2 exhibited significant inactivation (50%) and desensitization. The light sensitivity of Gt_CCR4 was higher (EC50 = 0.13 mW/mm2) than that of Cr_ChR2 (EC50 = 0.80 mW/mm2) while the channel open life time (photocycle speed) was in the same range as that of Cr_ChR2 (25~30 ms for Gt_CCR4 and 10~15 ms for Cr_ChR2). This observation implies that Gt_CCR4 enables optical neuronal spiking with weak light in high temporal resolution when applied in neuroscience. Furthermore, we demonstrated high Na+ selectivity of Gt_CCR4 in which the selectivity ratio for Na+ was 37-fold larger than that for Cr_ChR2, which primarily conducts H+. On the other hand, Gt_CCR4 conducted almost no H+ and no Ca2+ under physiological conditions. These results suggest that ion selectivity in Gt_CCR4 is distinct from that in Cr_ChR2. In addition, a unique red-absorbing and stable intermediate in the photocycle was observed, indicating a photochromic property of Gt_CCR4.
Highlights
Microbial-type rhodopsins are made up of seven or eight transmembrane helices with a covalently bound all-trans retinal as the chromophore [1]
These differences in the molecular mechanism place the cryptophyte CCR in a new family of channelrhodopsins, which we described as “DTD channelrhodopsins” or “BR-like cation channelrhodopsins” [29,30]
We transiently expressed Gt_CCR4 and Cr_ChR2 in ND7/23 cells by a conventional transfection method (FuGENE). Expression of these channels was visualized by tagged-GFP or Venus fluorescence
Summary
Microbial-type rhodopsins are made up of seven or eight transmembrane helices with a covalently bound all-trans retinal as the chromophore [1]. They are found in archaea, bacteria, eukaryota (such as fungi and algae) and viruses, and are physiologically responsible for energy production and the phototaxis reaction. Molecular functions of microbial rhodopsin involve ion transporters, sensors and light-regulated enzymes. As for ion-transporting rhodopsins, they are divided into ion-pumps and channels. Bacteriorhodopsin (BR) was the first identified outward directed proton-pumping rhodopsin [2]. The discovery of a Cl− pump, an Na+ pump and inward-directed proton pumps has been even until recently [3,4,5,6]. Structure-based and spectroscopic studies, when combined with electrophysiology and molecular dynamics studies, revealed the detailed molecular mechanism of bacteriorhodopsin and other pumps
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
