Molecular properties of a DTD channelrhodopsin from <i>Guillardia theta</i>

Abstract

Microbial rhodopsins are membrane proteins found widely in archaea, eubacteria and eukaryotes (fungal and algal species). They have various functions, such as light-driven ion pumps, light-gated ion channels, light sensors and light-activated enzymes. A light-driven proton pump bacteriorhodopsin (BR) contains a DTD motif at positions 85, 89, and 96, which is unique to archaeal proton pumps. Recently, channelrhodopsins (ChRs) containing the DTD motif, whose sequential identity is ~20% similar to BR and to cation ChRs in Chlamydomonas reinhardtii (CrCCRs), were found. While extensive studies on ChRs have been performed with CrCCR2, the molecular properties of DTD ChRs remain an intrigue. In this paper, we studied a DTD rhodopsin from G. theta (GtCCR4) using electrophysiological measurements, flash photolysis, and low-temperature difference FTIR spectroscopy. Electrophysiological measurements clearly showed that GtCCR4 functions as a light-gated cation channel, similar to other G. theta DTD ChRs (GtCCR1-3). Light-driven proton pump activity was also suggested for GtCCR4. Both electrophysiological and flash photolysis experiments showed that channel closing occurs upon reprotonation of the Schiff base, suggesting that the dynamics of retinal and channels are tightly coupled in GtCCR4. From Fourier transform infrared (FTIR) spectroscopy at 77 K, we found that the primary reaction is an all-trans to a 13-cis photoisomerization, like other microbial rhodopsins, although perturbations in the secondary structure were much smaller in GtCCR4 than in CrCCR2.

MerMAIDs: a family of metagenomically discovered marine anion-conducting and intensely desensitizing channelrhodopsins

“…As already shown, replacement of pore-lining glutamates with positively charged or neutral amino acids can mediate anion selectivity in originally cation-conducting chlorophyte CCRs [18][19][20][21][22] . Nevertheless, cryptophyte CCRs were shown to conduct cations although lacking typical glutamate motives by operating with an alternative mode more related to light-driven rhodopsin ion pumps [32][33][34] . To examine the MerMAIDs function and ion selectivity, we expressed them in human embryonic kidney (HEK) cells and performed whole-cell voltage-clamp experiments at 1-day posttransfection.…”

Ion Channel Properties of a Cation Channelrhodopsin, Gt_CCR4

“…In Cr_ChR2, D156 in TM4 provides the proton [35]. We demonstrated, using an FTIR study, that the secondary structural change in the primary reaction was much smaller than in Cr_ChR2 [30]. 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].…”

“…A novel cation channelrhodopsin family was reported in 2016 and 2017 from Guillardia theta, namely Gt_CCR1-4 [29,30]. These cation channelrhodopsins (CCRs) from cryptophyte algae are more homologous to haloarchaeal rhodopsins, such as proton pumping bacteriorhodopsin, than to chlorophyte CCRs, including Cr_ChR2.…”

Bacteriorhodopsin-like channelrhodopsins: Alternative mechanism for control of cation conductance

“…1). Surprisingly, when these "BR-like" cryptophyte rhodopsins were tested by expression in cultured animal cells and patch clamp analysis, they demonstrated nonspecific cation channel activity similar to that of chlorophyte CCRs (20,22). However, electrophysiological data obtained under continuous light stimulation, as in the above cited studies, do not allow precise deconvolution of photocurrent components and may lead to incorrect interpretation of their nature.…”